Drug regulated transgene expression

ABSTRACT

The present invention provides nucleic acids, vectors, host cells, methods and compositions to confer and/or augment immune responses mediated by cellular immunotherapy, such as by adoptively transferring CD8+ central memory T cells or combinations of central memory T cells with CD4+ T cells that are genetically modified to express a chimeric receptor under the control of an inducible promoter. In some alternatives the genetically modified host cell comprises a nucleic acid comprising a polynucleotide coding for a chimeric antigen receptor comprising a ligand binding domain, a polynucleotide comprising a spacer region, a polynucleotide comprising a transmembrane domain, and a polynucleotide comprising an intracellular signaling domain under the control of a drug inducible promoter. Controlling the expression of the chimeric receptor provides for the ability to turn expression on and off depending on the status of the patient. Pharmaceutical formulations produced by the method, and methods of using the same, are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/302,415 filed Oct. 6, 2016 which is a U.S. National Phase Applicationof PCT International Application Number PCT/US2015/024947, filed on Apr.8, 2015, designating the United States of America and published in theEnglish language, which claims the benefit of priority to U.S. Prov.App. No. 62/058,973, filed Oct. 2, 2014, U.S. Prov. App. No. 61/977,751,filed Apr. 10, 2014, U.S. Prov. App. No. 61/986,479, filed Apr. 30,2014, U.S. Prov. App. No. 62/089,730 filed Dec. 9, 2014, U.S. Prov. App.No. 62/090,845, filed Dec. 11, 2014, and U.S. Prov. App. No. 62/088,363,filed Dec. 5, 2014. The entire disclosures of the aforementionedapplications are expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSCRI053C1SEQLISTING.TXT, created Feb. 20, 2019, which is approximately72 Kb in size. The information in the electronic format of the SequenceListing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The adoptive transfer of human T lymphocytes that are engineered by genetransfer to express chimeric antigen receptors (CARs) specific forsurface molecules expressed on tumor cells has the potential toeffectively treat cancer. Chimeric receptors are synthetic receptorsthat include an extracellular ligand binding domain, most commonly asingle chain variable fragment of a monoclonal antibody (scFv) linked tointracellular signaling components, most commonly CD3ζ alone or combinedwith one or more costimulatory domains. Much of the research in thedesign of chimeric receptors has focused on defining scFvs and otherligand binding elements that target malignant cells without causingserious toxicity to essential normal tissues, and on defining theoptimal composition of intracellular signaling modules to activate Tcell effector functions.

Although, T cell (CAR-T) adoptive therapy clinical trials (chimericantigen receptor expressing T cells) are demonstrating potent anti-tumoractivity, it is apparent that significant toxicities can arise, forexample, engraftment-induced cytokine storm, tumor lysis syndromes andongoing B cell cytopenias, each of which are attributable to unregulatedfunctional outputs of constitutively expressed CARs. Such toxicities canin some context threaten to limit the applicability of CAR-T celladoptive therapy. Clinical trials using transgene-modified adoptive Tcell immunotherapies have only tested T cells that constitutivelyexpress the transgene, or are always in the “ON” state, contributing inlarge part to transgene associated side-effects. Suicide gene-mediatedelimination of CAR-T cells can ameliorate such toxicities; however, thisapproach risks premature attenuation of anti-tumor activity andsignificantly impacts curative potential.

Current small molecule-regulated transgene expression technologies relyon a variety of drug inputs including macrolides, ecdysones andrapamycin analogs. Clinical applicability of these systems is limiteddue to toxic off target effects, unfavorable biodistribution andpharmacodynamics profiles, limited output dynamic range, and/or limitedavailability as FDA-approved commercially available pharmaceuticals.Furthermore, many of these systems use chimeric transcriptionalregulators built from xenogeneic components, thus introducing thecomplication of immunogenicity when applying these systems to humantherapeutics.

There is a need to identify methods for determining elements of chimericreceptor design that are important for therapeutic activity and cellpopulations to genetically modify and adoptively transfer that willprovide enhanced survival and efficacy in vivo while minimizing adverseside effects. There is also a need for expression systems and methodsfor modulating cells for use in cell therapy, such as for modulatingexpression of recombinant antigen receptors such as CARs and/or othermolecules expressed by such cells, such as to improve therapeuticactivity, enhanced survival and/or efficacy in vivo and/or minimizeadverse side effects.

SUMMARY OF THE INVENTION

One aspect of the disclosure includes a genetic system to deliverdrug-regulated transgene expression in cells. In an alternative,regulated transgene expression is targeted to cells, such as lymphocytesdesigned for use in adoptive immunotherapy. This system providesrigorous safety attributes to chimeric antigen receptor (CAR) redirectedadoptive therapeutic strategies without sacrificing curative intent thatpermits real-time clinician control of CAR expression in vivo. Byengineering vectors that enable drug responsive transcriptional controlof CAR expression, the activity of CARs and other cell mediators can beturned “ON” and “OFF” in vivo, based on a clinician prescribedpharmaceutical drug input that exhibits clinically permissivepharmacokinetics, tissue distribution, and partitioning between theextracellular space and cytosol of lymphocytes. The genetic systemprovides for drug regulated transgene expression to enforce a functional“OFF” state in the absence of the drug and a functional “ON” statetransgene expression in the presence of the drug.

One alternative of such a drug is tamoxifen. Tamoxifen is an estrogenantagonist/partial agonist that is an FDA-approved and commerciallyavailable drug. It is taken orally and can be administered on a dailybasis over an extended period of time. Tamoxifen has a proven safetyrecord, favorable pharmacokinetic profile, excellent tissue distributionand a low partition coefficient between the extracellular space andcytosol. Other drugs can be selected based on safety record, favorablepharmacokinetic profile, and excellent tissue distribution, a lowpartition coefficient between the extracellular space and cytosol,and/or low toxicities.

In some alternatives, the system employs a synthetic transcriptionalregulator, which, in the presence of tamoxifen, binds a syntheticpromoter upstream of a transgene to induce expression. The tamoxifenregulated transcription factor (“TamR-tf”, also designated “HEA3”) is achimeric transcription factor composed of human subunits including theN-terminal DNA binding domain of Hepatocyte Nuclear Factor 1-alpha(HNF-1α) fused in frame to the mutant tamoxifen-specific ligand bindingdomain of the estrogen receptor ligand binding domain (ER-LBD), that isin turn fused to the p65 activation domain of NF-κB (p65). An exemplaryamino acid sequence is provided in FIGS. 9A, 9B, and 9C and isidentified as SEQ ID NO: 40. The mutant tamoxifen-specific ligandbinding domain of the estrogen receptor ligand binding domain (ER-LBD)is found at amino acids 282-595 of the TamR-tf and has a mutation atposition 521. The p65 activation domain of NF-κB (p65) is found at aminoacids 596-862 of SEQ ID NO: 40. Further changes can be made to thetranscriptional activator to increase the properties of thetranscription factor including, without limitation, altering one or moreamino acids in the estrogen receptor ligand binding domain to increasethe affinity of the factor for estrogen analogs and altering one or moreamino acids in the p65 transactivating domain.

In the absence of tamoxifen, TamR-tf is excluded from the nucleus bybinding of cytosolic heat-shock protein 90 (HSP90) to the tamoxifenbinding active site and transgene expression is in the “OFF” state.Nanomolar concentrations of cytosolic tamoxifen actively out competesHSP90 for ER-LBD binding, resulting in TamR-tf translocation to thenucleus. Upon nuclear translocation, TamR-tf is readily available tobind its restricted synthetic promoter (e.g. 7xHBD/EF1αp). In thepresence of tamoxifen, binding of TamR-tf to 7xHBD/EF1αp promoterinduces the “ON” state of transgene expression. In some alternatives,this transcriptional regulator can be modified to provide for a varyinglevel of control of transgene expression. Amino acid substitutions inthe LBD of TamR-tf permit selective responsiveness to tamoxifen and itsmetabolites, where 4-hydroxy tamoxifen (4-OHT) is the mostpharmacologically active metabolite, in regards to TamR-tf activity,while lacking interaction with endogenous estrogen.

In one aspect, the present disclosure relates to methods andcompositions to confer and/or augment immune responses mediated bycellular immunotherapy, such as, by adoptively transferringtumor-specific, genetically modified subsets of CD8+ or CD4+ T cellsalone, or in combination. The disclosure provides for chimeric receptornucleic acids, and vectors and host cells including such nucleic acids.The nucleic acid sequence that encodes the chimeric receptor linkstogether a number of modular components that can be excised and replacedwith other components in order to customize the chimeric receptor forefficient T cell activation and recognition of a specific targetmolecule or an epitope on the target molecule and provide for regulatedtranscription as described herein.

In some alternatives, a system for inducible expression of a chimericantigen receptor comprises: a first nucleic acid comprising a firstpromoter inducible by a drug, wherein the first nucleic acid is operablylinked to a polynucleotide coding for a chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; apolynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the spacer is optimized for increasedT cell proliferation and/or cytokine production in response to theligand as compared to a reference chimeric receptor. In somealternatives, the second promoter is constitutive or inducible.

In some alternatives, a system for inducible expression of a chimericantigen receptor comprises: a first nucleic acid comprising a firstpromoter inducible by a drug, wherein the first nucleic acid is operablylinked to a polynucleotide coding for a cytokine, a chemokine, apolypeptide that regulates apoptosis and/or a polypeptide that modulatescheckpoint signaling; and a second nucleic acid comprising a secondpromoter operably linked to a nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the secondpromoter is constitutive or inducible. In an exemplary alternative, thesecond nucleic acid further comprises a polynucleotide coding for achimeric antigen receptor, under the control of a constitutive promoter.In some alternatives, the drug is tamoxifen and/or its metabolites.

In another aspect, the present disclosure provides compositions toconfer and/or augment immune responses mediated by cellularimmunotherapy, such as by adoptively transferring tumor-specific, subsetspecific genetically modified CD4+ T cells, wherein the CD4+ T cellsconfer and/or augment the ability of CD8+ T cells to sustain anti-tumorreactivity and increase and/or maximize tumor-specific proliferation. Insome alternatives, the CD4+ cells are genetically modified to express achimeric receptor nucleic acid and/or chimeric receptor polypeptideunder the control of a regulated promoter, as described herein.

In another aspect, the present disclosure provides compositions toconfer and/or augment immune responses mediated by cellularimmunotherapy, such as by adoptively transferring tumor-specific, subsetspecific genetically modified T cells. In some alternatives, the cellsare precursor T cells. In some alternatives, the cells are hematopoieticstem cells. In some alternatives, the cells are CD8+ T cells. In somealternatives, the CD8+ T cells express a chimeric receptor nucleic acidand/or chimeric receptor polypeptide under the control of a regulatedpromoter, as described herein.

Some alternatives concern methods of performing cellular immunotherapyin a subject having a disease or disorder by administering to thesubject a genetically modified T lymphocyte cell preparation thatprovides a cellular immune response and administering a drug thatinduces a transgene in the genetically modified T lymphocyte cells. Insome alternatives, the genetically modified T lymphocyte cellpreparation comprises precursor T cells. In some alternatives, thegenetically modified T lymphocyte cell preparation compriseshematopoietic stem cells. In some alternatives, the genetically modifiedCD8+ and genetically modified CD4+ cell population are co-administered.In some alternatives, the drug is tamoxifen and/or its metabolites. Insome alternatives, the T cells are autologous or allogeneic T cells.Various modifications of the above method are possible. For example, thechimeric receptor that is expressed by the CD4+ T cell and the CD8+ Tcell can be the same or different.

In some alternatives, a system for inducible expression of a chimericantigen receptor is provided, wherein the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora chimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the second promoter is inducible. Insome alternatives, the second promoter is constitutive. In somealternatives, the first promoter comprises a nucleic acid sequence ofSEQ ID NO: 41. In some alternatives, the second promoter is aconstitutive promoter. In some alternatives, the second promoter is theEF1αp. In some alternatives, the transcriptional activator comprises asequence of SEQ ID NO: 40. In some alternatives, the first nucleic acidfurther comprises a first vector and the second nucleic acid furthercomprises a second vector. In some alternatives, both vectors arepackaged in a viral vector. In some alternatives, the viral vector is alentivirus. In some alternatives, the first and second nucleic acidcomprise a vector. In some alternatives, the first nucleic acid furthercomprises a nucleic acid sequence coding for a selectable marker. Insome alternatives, the second nucleic acid further comprises a nucleicacid coding for a selectable marker. In some alternatives, the spacer isoptimized for increased T cell proliferation and/or cytokine productionin response to the ligand as compared to a reference chimeric receptor.

In some alternatives, a system for inducible expression of a chimericantigen receptor is provided, wherein the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora chimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the second promoter is constitutive orinducible. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for a chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the drug is tamoxifen and/or its metabolites. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment. In some alternatives, the ligand binding domain issingle chain variable fragment. In some alternatives, the tumor specificmolecule is CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3,mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinationsthereof. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor. In some alternatives, thesecond promoter is inducible. In some alternatives, the second promoteris constitutive.

In some alternatives, a chimeric receptor polypeptide is provided,wherein the chimeric receptor polypetide is coded for by a system. Insome alternatives, the system comprises a) a first nucleic acidcomprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the second promoter is constitutive orinducible. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesecond promoter is an inducible promoter. In some alternatives, thespacer is optimized for increased T cell proliferation and/or cytokineproduction in response to the ligand as compared to a reference chimericreceptor.

In some alternatives, a system for inducible expression of chimericantigen receptor is provided, wherein the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora cytokine, a chemokine receptor, a polypeptide that regulatesapoptosis, or a polypeptide that modulates checkpoint signaling and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the second promoter is constitutive orinducible. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for a chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment, preferably a binding domain thereof. In somealternatives, the tumor specific molecule is CD19, CD20, CD22, CD23,CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2,GD-2, or MAGE A3 TCR or combinations thereof. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling; and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the secondpromoter is constitutive or inducible. In some alternatives, the drug istamoxifen and/or its metabolites. In some alternatives, the secondnucleic acid further comprises a polynucleotide coding for chimericantigen receptor comprising a ligand binding domain, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment, preferably a binding fragment thereof. In somealternatives, the ligand binding domain is single chain variablefragment. In some alternatives, the tumor specific molecule is CD19,CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor.

In some alternatives, a host cell is provided, wherein the host cellcomprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand is a tumor specific molecule, viral molecule,or any other molecule expressed on a target cell population, wherein theligand can elicit recognition, modulation, inhibition, and/orelimination by a lymphocyte; a polynucleotide coding for a polypeptidespacer, wherein the spacer is optimized; a polynucleotide coding for atransmembrane domain; and d) a polynucleotide coding for anintracellular signaling domain; and b) a second nucleic acid comprisinga second promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the second promoter is constitutive or inducible. In somealternatives, the drug is tamoxifen and/or its metabolites. In somealternatives, the first promoter comprises a nucleic acid sequence ofSEQ ID NO: 41. In some alternatives, the second promoter is aconstitutive promoter. In some alternatives, the second promoter is theEF1αp. In some alternatives, the transcriptional activator comprises asequence of SEQ ID NO: 40. In some alternatives, the first nucleic acidfurther comprises a first vector and the second nucleic acid furthercomprises a second vector. In some alternatives, both vectors arepackaged in a viral vector. In some alternatives, the viral vector is alentivirus. In some alternatives, the first and second nucleic acidcomprise a vector. In some alternatives, the first nucleic acid furthercomprises a nucleic acid sequence coding for a selectable marker. Insome alternatives, the second nucleic acid further comprises a nucleicacid coding for a selectable marker. In some alternatives, the secondnucleic acid further comprises a polynucleotide coding for a chimericantigen receptor comprising a ligand binding domain, wherein the ligandbinding domain is specific for a ligand, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment, preferably a binding fragmentthereof. In some alternatives, the tumor specific molecule is CD19,CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter inducible by a drug, wherein the first nucleic acid isoperably linked to a polynucleotide coding for a cytokine, a chemokinereceptor, a polypeptide that regulates apoptosis, or a polypeptide thatmodulates checkpoint signaling; and b) a second nucleic acid comprisinga second promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the second promoter is constitutive or inducible. In somealternatives, the second nucleic acid further comprises a polynucleotidecoding for chimeric antigen receptor comprising a ligand binding domain,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; and d) a polynucleotide coding for anintracellular signaling domain. In some alternatives, the first promoteris in opposite orientation to the second promoter. In some alternatives,the ligand binding domain is an antibody fragment, preferably a bindingfragment thereof. In some alternatives, the ligand binding domain issingle chain variable fragment. In some alternatives, the tumor specificmolecule is CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3,mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinationsthereof. In some alternatives, the host cell is a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cells. In some alternatives, the host cell is a CD4+ Thelper lymphocyte cell that is selected from the group consisting ofnaïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ Tcells, and bulk CD4+ T cells. In some alternatives, the spacer isoptimized for increased T cell proliferation and/or cytokine productionin response to the ligand as compared to a reference chimeric receptor.In some alternatives, the host cell is precursor T cell. In somealternatives, the precursor T cell is a hematopoietic stem cell.

In some alternatives, a composition is provided, wherein the compositioncomprises a host cell in a pharmaceutically acceptable excipient. Insome alternatives, the host cell comprises a system. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter inducible by a drug, wherein the first nucleic acid isoperably linked to a polynucleotide coding for a chimeric antigenreceptor comprising a ligand binding domain, wherein the ligand is atumor specific molecule, viral molecule, or any other molecule expressedon a target cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the second promoter is constitutive orinducible. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand is a tumor specific molecule, viral molecule, or any othermolecule expressed on a target cell population, wherein the ligand canelicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment. In some alternatives, the tumorspecific molecule is CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7,EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR orcombinations thereof. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a cytokine, a chemokine receptor, a polypeptide thatregulates apoptosis, or a polypeptide that modulates checkpointsignaling; and b) a second nucleic acid comprising a second promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the second promoter isconstitutive or inducible. In some alternatives, the second nucleic acidfurther comprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment, preferably a binding fragment thereof. In somealternatives, the ligand binding domain is single chain variablefragment. In some alternatives, the tumor specific molecule is CD19,CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the host cell is precursor T cell. In some alternatives,the precursor T cell is a hematopoietic stem cell. In some alternatives,the host cell is a CD8+ T cytotoxic lymphocyte cell selected from thegroup consisting of naïve CD8+ T cells, central memory CD8+ T cells,effector memory CD8+ T cells and bulk CD8+ T cells. In somealternatives, the host cell is a CD4+ T helper lymphocyte cell that isselected from the group consisting of naïve CD4+ T cells, central memoryCD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. Insome alternatives, the composition comprises a host cell wherein thehost cell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells and further comprises anotherhost cell wherein the host cell is a CD4+ T helper lymphocyte cell thatis selected from the group consisting of naïve CD4+ T cells, centralmemory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ Tcells. In some alternatives, the host cell is precursor T cell. In somealternatives, the precursor T cell is a hematopoietic stem cell. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor. In some alternatives, the compositioncomprises a host cell wherein the host cell is a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cells or the host cell is a CD4+ T helper lymphocyte cellthat is selected from the group consisting of naïve CD4+ T cells,central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+T cells and a second host cell, wherein the second host cell is aprecursor T cell or a hematopoietic stem cell.

In some alternatives, an in vitro method for preparing a host cell isprovided wherein the method comprises a) providing a system and b)introducing the system into a separate isolated T lymphocyte populationand expanding each T lymphocyte population in vitro. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter inducible by a drug, wherein the first nucleic acid isoperably linked to a polynucleotide coding for a chimeric antigenreceptor comprising a ligand binding domain, wherein the ligand bindingdomain is specific for a ligand, wherein the ligand is a tumor specificmolecule, viral molecule, or any other molecule expressed on a targetcell population, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; apolynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the second promoter is constitutive orinducible. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the second promoter is constitutiveor inducible. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment, preferably a binding fragment thereof. In somealternatives, the tumor specific molecule is CD19, CD20, CD22, CD23,CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2,GD-2, or MAGE A3 TCR or combinations thereof. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling; and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the secondpromoter is constitutive or inducible. In some alternatives, the secondnucleic acid further comprises a polynucleotide coding for chimericantigen receptor comprising a ligand binding domain, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment, preferably a binding fragment thereof. In somealternatives, the ligand binding domain is single chain variablefragment. In some alternatives, the tumor specific molecule is CD19,CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, wherein the T lymphocytes are expanded, the method furthercomprises culturing the cells in the presence of anti-CD3 and/or antiCD28, and at least one homeostatic cytokine until the cells expandsufficiently for use as a cell infusion. In some alternatives, thelymphocyte is CD8+ or CD4+. In some alternatives, the spacer isoptimized for increased T cell proliferation and/or cytokine productionin response to the ligand as compared to a reference chimeric receptor.In some alternatives, the host cell is precursor T cell. In somealternatives, the precursor T cell is a hematopoietic stem cell.

In some alternatives, a use of a host cell or a composition incombination with a drug that induces expression of a transgene in thehost cell or composition for the treatment of cancer or a viralinfection is provided. In some alternatives, the host cell comprises asystem. In some alternatives, the system comprises a) a first nucleicacid comprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the second promoter is constitutive orinducible. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment. In some alternatives, the tumorspecific molecule is CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7,EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR orcombinations thereof. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a cytokine, a chemokine receptor, a polypeptide thatregulates apoptosis, or a polypeptide that modulates checkpointsignaling; and b) a second nucleic acid comprising a second promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the second promoter isconstitutive or inducible. In some alternatives, the second nucleic acidfurther comprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment, preferably a binding fragmentthereof. In some alternatives, the ligand binding domain is single chainvariable fragment. In some alternatives, the tumor specific molecule isCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the host cell is a CD8+ T cytotoxic lymphocyte cellselected from the group consisting of naïve CD8+ T cells, central memoryCD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. Insome alternatives, the host cell is a CD4+ T helper lymphocyte cell thatis selected from the group consisting of naïve CD4+ T cells, centralmemory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ Tcells. In some alternatives, the cells are precursor T cells. In somealternatives, the cells are hematopoietic stem cells. In somealternatives, the composition comprises a host cell in apharmaceutically acceptable excipient. In some alternatives, the hostcell comprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; and b) a second nucleic acid comprising asecond promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the second promoter is constitutive or inducible. In somealternatives, the drug is tamoxifen and/or its metabolites. In somealternatives, the first promoter comprises a nucleic acid sequence ofSEQ ID NO: 41. In some alternatives, the second promoter is aconstitutive promoter. In some alternatives, the second promoter is theEF1αp. In some alternatives, the transcriptional activator comprises asequence of SEQ ID NO: 40. In some alternatives, the first nucleic acidfurther comprises a first vector and the second nucleic acid furthercomprises a second vector. In some alternatives, both vectors arepackaged in a viral vector. In some alternatives, the viral vector is alentivirus. In some alternatives, the first and second nucleic acidcomprise a vector. In some alternatives, the first nucleic acid furthercomprises a nucleic acid sequence coding for a selectable marker. Insome alternatives, the second nucleic acid further comprises a nucleicacid coding for a selectable marker. In some alternatives, the secondnucleic acid further comprises a polynucleotide coding for a chimericantigen receptor comprising a ligand binding domain, wherein the ligandbinding domain is specific for a ligand, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment, preferably a binding fragmentthereof. In some alternatives, the tumor specific molecule is CD19,CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter inducible by a drug, wherein the first nucleic acid isoperably linked to a polynucleotide coding for a cytokine, a chemokinereceptor, a polypeptide that regulates apoptosis, or a polypeptide thatmodulates checkpoint signaling; and b) a second nucleic acid comprisinga second promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the second promoter is constitutive or inducible. In somealternatives, the second nucleic acid further comprises a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand is a tumor specific molecule, viral molecule,or any other molecule expressed on a target cell population, wherein theligand can elicit recognition, modulation, inhibition, and/orelimination by a lymphocyte; a polynucleotide coding for a polypeptidespacer, wherein the spacer is optimized; and d) a polynucleotide codingfor an intracellular signaling domain. In some alternatives, the firstpromoter is in opposite orientation to the second promoter. In somealternatives, the ligand binding domain is an antibody fragment,preferably a binding fragment thereof. In some alternatives, the ligandbinding domain is single chain variable fragment. In some alternatives,the tumor specific molecule is CD19, CD20, CD22, CD23, CD123, CS-1,ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3TCR or combinations thereof. In some alternatives, the host cell is aCD8+ T cytotoxic lymphocyte cell selected from the group consisting ofnaïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ Tcells and bulk CD8+ T cells. In some alternatives, the host cell is aCD4+ T helper lymphocyte cell that is selected from the group consistingof naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+T cells, and bulk CD4+ T cells. In some alternatives, the compositioncomprises a host cell wherein the host cell is a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cells and another host cell wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting ofnaïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ Tcells, and bulk CD4+ T cells. In some alternatives, the cancer is asolid tumor or hematologic malignancy. In some alternatives, the solidtumor is selected from the group consisting of a breast cancer, braincancer, lung cancer, colon cancer, renal cancer, pancreatic cancer,prostate cancer, and ovarian cancer. In some alternatives, the spacer isoptimized for increased T cell proliferation and/or cytokine productionin response to the ligand as compared to a reference chimeric receptor.In some alternatives, the host cell is precursor T cell. In somealternatives, the precursor T cell is a hematopoietic stem cell. In somealternatives, the host cell is precursor T cell. In some alternatives,the precursor T cell is a hematopoietic stem cell. In some alternatives,the spacer is optimized for increased T cell proliferation and/orcytokine production in response to the ligand as compared to a referencechimeric receptor. In some alternatives, the composition comprises ahost cell wherein the host cell is a CD8+ T cytotoxic lymphocyte cellselected from the group consisting of naïve CD8+ T cells, central memoryCD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells or thehost cell is a CD4+ T helper lymphocyte cell that is selected from thegroup consisting of naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells, and bulk CD4+ T cells and a second hostcell, wherein the second host cell is a precursor T cell or ahematopoietic stem cell.

In some alternatives, a method of performing cellular immunotherapy in asubject having cancer or a viral infection is provided wherein themethod comprises administering a composition or a host cell to thesubject and administering a drug that induces expression of a transgenein the composition or the host cells. In some alternatives, the hostcell comprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain; and b) a second nucleic acid comprising a second promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the second promoter isconstitutive or inducible. In some alternatives, the drug is tamoxifenand/or its metabolites. In some alternatives, the first promotercomprises a nucleic acid sequence of SEQ ID NO: 41. In somealternatives, the second promoter is a constitutive promoter. In somealternatives, the second promoter is the EF1αp. In some alternatives,the transcriptional activator comprises a sequence of SEQ ID NO: 40. Insome alternatives, the first nucleic acid further comprises a firstvector and the second nucleic acid further comprises a second vector. Insome alternatives, both vectors are packaged in a viral vector. In somealternatives, the viral vector is a lentivirus. In some alternatives,the first and second nucleic acid comprise a vector. In somealternatives, the first nucleic acid further comprises a nucleic acidsequence coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a nucleic acid coding for aselectable marker. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for a chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment, preferably a binding fragment thereof. In somealternatives, the tumor specific molecule is CD19, CD20, CD22, CD23,CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2,GD-2, or MAGE A3 TCR or combinations thereof. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling; and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the secondpromoter is constitutive or inducible. In some alternatives, the secondnucleic acid further comprises a polynucleotide coding for a chimericantigen receptor comprising a ligand binding domain, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment, preferably a binding fragment thereof. In somealternatives, the ligand binding domain is single chain variablefragment. In some alternatives, the tumor specific molecule is CD19,CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the host cell is a CD8+ T cytotoxic lymphocyte cellselected from the group consisting of naïve CD8+ T cells, central memoryCD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. Insome alternatives, the host cell is a CD4+ T helper lymphocyte cell thatis selected from the group consisting of naïve CD4+ T cells, centralmemory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ Tcells. In some alternatives, the composition comprises a host cell in apharmaceutically acceptable excipient. In some alternatives, the hostcell comprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; and b) a second nucleic acid comprising asecond promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the second promoter is constitutive or inducible. In somealternatives, the drug is tamoxifen and/or its metabolites. In somealternatives, the first promoter comprises a nucleic acid sequence ofSEQ ID NO: 41. In some alternatives, the second promoter is aconstitutive promoter. In some alternatives, the second promoter is theEF1αp. In some alternatives, the transcriptional activator comprises asequence of SEQ ID NO: 40. In some alternatives, the first nucleic acidfurther comprises a first vector and the second nucleic acid furthercomprises a second vector. In some alternatives, both vectors arepackaged in a viral vector. In some alternatives, the viral vector is alentivirus. In some alternatives, the first and second nucleic acidcomprise a vector. In some alternatives, the first nucleic acid furthercomprises a nucleic acid sequence coding for a selectable marker. Insome alternatives, the second nucleic acid further comprises a nucleicacid coding for a selectable marker. In some alternatives, the secondnucleic acid further comprises a polynucleotide coding for a chimericantigen receptor comprising a ligand binding domain, wherein the ligandbinding domain is specific for a ligand, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment, preferably a binding fragmentthereof. In some alternatives, the tumor specific molecule is CD19,CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter inducible by a drug, wherein the first nucleic acid isoperably linked to a polynucleotide coding for a cytokine, a chemokinereceptor, a polypeptide that regulates apoptosis, or a polypeptide thatmodulates checkpoint signaling; and b) a second nucleic acid comprisinga second promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the second promoter is constitutive or inducible. In somealternatives, the second nucleic acid further comprises a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand is a tumor specific molecule, viral molecule,or any other molecule expressed on a target cell population, wherein theligand can elicit recognition, modulation, inhibition, and/orelimination by a lymphocyte; a polynucleotide coding for a polypeptidespacer, wherein the spacer is optimized; and d) a polynucleotide codingfor an intracellular signaling domain. In some alternatives, the firstpromoter is in opposite orientation to the second promoter. In somealternatives, the ligand binding domain is an antibody fragment,preferably a binding fragment thereof. In some alternatives, the ligandbinding domain is single chain variable fragment. In some alternatives,the tumor specific molecule is CD19, CD20, CD22, CD23, CD123, CS-1,ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, or MAGE A3TCR or combinations thereof. In some alternatives, the host cell is aCD8+ T cytotoxic lymphocyte cell selected from the group consisting ofnaïve CD8+ T cells, central memory CD8+ T cells, effector memory CD8+ Tcells and bulk CD8+ T cells. In some alternatives, the host cell is aCD4+ T helper lymphocyte cell that is selected from the group consistingof naïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+T cells, and bulk CD4+ T cells. In some alternatives, the compositioncomprises a host cell wherein the host cell is a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cells and another host cell wherein the host cell is a CD4+T helper lymphocyte cell that is selected from the group consisting ofnaïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ Tcells, and bulk CD4+ T cells. In some alternatives, the host cell isprecursor T cell. In some alternatives, the precursor T cell is ahematopoietic stem cell. In some alternatives, the spacer is optimizedfor increased T cell proliferation and/or cytokine production inresponse to the ligand as compared to a reference chimeric receptor. Insome alternatives, the composition comprises a host cell wherein thehost cell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells or the host cell is a CD4+ Thelper lymphocyte cell that is selected from the group consisting ofnaïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ Tcells, and bulk CD4+ T cells and a second host cell, wherein the secondhost cell is a precursor T cell or a hematopoietic stem cell. In somealternatives, the cancer is selected from a solid tumor or hematologicmalignancy. In some alternatives, the solid tumor is selected from thegroup consisting of a breast cancer, brain cancer, lung cancer, coloncancer, renal cancer, pancreatic cancer, prostate cancer, and ovariancancer. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor. In some alternatives, thehost cell is a precursor T cell. In some alternatives, the precursor Tcell is a hematopoietic stem cell. In some alternatives, the isolated Tlymphocyte population comprises precursor T cells. In some alternatives,the precursor T cells are hematopoietic stem cells. In somealternatives, the administering of the drug is performed afteradministering of the composition or host cells, wherein administering isperformed 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2weeks, 4 weeks or two months, or any time in between any two values oftime listed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the expression of ZsGreen and EGFRt as determined by flowcytometry in Jurkat cells transduced with a dual plasmid lentiviralconstruct including constructs A and B in the presence or absence of 4hydroxy tamoxifen (4OHT). The results show the expression of EGFRt inthe presence or absence of 4-OHT indicating the cells carry construct A.The results also show that the cells carry construct B as expression ofZsGreen is induced in the presence of 4-OHT. FIG. 1B shows construct A,which comprises the constitutive promoter EF1αp linked to TamR-tf (HEA3)linked to EGFRt, and construct B comprising a synthetic promoter7xHBD/mE1b linked to a polynucleotide coding for ZsGreen.

FIG. 2 shows the expression of ZsGreen in transduced Jurkat cellscontacted with different doses of 4-OHT ranging from 50 to 1000 nM.

FIG. 3 shows the on and off rate kinetics of expression of ZsGreen intransduced Jurkat cells with a single 48 hour treatment of 4-OHTfollowed by a washout (∘) and tranduced Jurkat cells with a 24 hourtreatment of 4-OHT followed by washout and then a re-stimulation with4-OHT at day 15 (▪).

FIG. 4A-D shows the expression of ZsGreen in CD4 central memory cellstransduced with dual package lentiviral constructs A and B. Cells weredivided into 3 treatment groups, 4OHT alone (FIG. 4A), 4OHT combinedwith CD3/CD28 bead co-treatment (FIG. 4B) or 4OHT alone for 48 hours,followed by addition of CD3/CD28 beads (FIG. 4C). Expression of ZsGreenwas monitored over 96 hours. FIG. 4D shows construct A, which comprisesthe constitutive promoter EF1αp linked to TamR-tf (HEA3) linked toEGFRt, and construct B comprising a synthetic promoter 7xHBD/mE1b linkedto a polynucleotide coding for ZsGreen.

FIGS. 5A-C shows the expression of ZsGreen in CD8 central memory cellstransduced with dual package lentiviral constructs A and B as shown inFIG. 4. Cells were divided into 3 treatment groups, 4OHT alone (FIG.5A), 4OHT combined with CD3/CD28 bead co-treatment (FIG. 5B) or 4OHTalone for 48 hours, followed by addition of CD3/CD28 beads (FIG. 5C).Expression of ZsGreen was monitored over 72 hours.

FIG. 6A shows expression of EGFRt and Her2t in Human Jurkat T cellstransduced with constructs A and B as shown in FIG. 6C. Expression ofEGFRt and Her2t were monitored in the presence or absence of 4OHT.Samples were stained with EGFRt-APC antibody and Herceptin-biotin,followed by SA-PE. FIG. 6B shows a western blot of parental Jurkat cellsand transduced CD19CAR transduced cells stained with mouse antiCD247which recognizes the intracellular CD3 zeta chain on CD19 CAR (about 48kDA), the endogenous CD3 zeta chain has migrates at 23 kDA. Theendogenous CD3 zeta chain was detected in all of the cells. The CD19 CARCD3 zeta chain was only detected in transduced Jurkat cells exposed to4-OHT. FIG. 6C shows construct A, which comprises the constitutivepromoter EF1αp linked to TamR-tf (HEA3) linked to EGFRt, and construct Bcomprising a synthetic promoter 7xHBD/mE1b linked to a polynucleotidecoding for CD19CAR linked to a polynucleotide coding for Her2t.

FIG. 7A shows expression of EGFRt and Her2t in Human Jurkat T cellstransduced with TamR CD19CAR LV including an additional selectivemarker, DHFRdm in the presence or absence of 4OHT. Expression of EGFRtand Her2t were monitored in the presence or absence of 4OHT. Sampleswere stained with EGFRt-APC antibody and Herceptin-biotin, followed bySA-PE. FIG. 7B shows a western blot of parental Jurkat cells andtransduced CD19CAR transduced cells stained with mouse antiCD247 whichrecognizes the intracellular CD3 zeta chain on CD19 CAR (about 48 kDA),the endogenous CD3 zeta chain has migrates at 23 kDA. The endogenous CD3zeta chain was detected in all of the cells. The CD19 CAR CD3 zeta chainwas only detected in transduced Jurkat cells exposed to 4-OHT. FIG. 7Cshows construct A, which comprises the constitutive promoter EF1αplinked to TamR-tf (HEA3) linked to EGFRt, and construct B comprising asynthetic promoter 7xHBD/mE1b linked to a polynucleotide coding forCD19CAR linked to a polynucleotide coding for Her2t linked to apolynucleotide coding for DHFRdm.

FIG. 8A shows expression of EGFRt and Her2t in Human CD4 central memoryT cells transduced with TamR CD19CAR LV including an additionalselective marker, DHFRdm in the presence or absence of 4OHT andantiCD3/CD28 beads. Expression of EGFRt and Her2t were monitored in thepresence or absence of 4OHT and in the presence or absence ofantiCD3/CD28 beads. Samples were stained with EGFRt-APC antibody andHerceptin-biotin, followed by SA-PE. FIG. 8B shows construct A, whichcomprises the constitutive promoter EF1αp linked to TamR-tf (HEA3)linked to EGFRt, and construct B comprising a synthetic promoter7xHBD/mE1b linked to a polynucleotide coding for CD19CAR linked to apolynucleotide coding for Her2t linked to a polynucleotide coding forDHFRdm.

FIG. 9A, FIG. 9B, and FIG. 9C depict a single HEA3 nucleotide sequence(SEQ ID NO:39) and amino acid sequence (SEQ ID NO:40).

FIG. 10A, FIG. 10B, and FIG. 10C depict a single HEA4 nucleotidesequence (SEQ ID NO:42) and amino acid sequence (SEQ ID NO:43).

FIG. 11 depicts a HER2t nucleotide sequence (SEQ ID NO:44) and aminoacid sequence (SEQ ID NO:45).

FIG. 12 depicts a DHFRdm nucleotide sequence (SEQ ID NO:46) and aminoacid sequence (SEQ ID NO:47).

DETAILED DESCRIPTION Definitions.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains.

“About” as used herein when referring to a measurable value is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue.

Antigen” or “Ag” as used herein refers to a molecule that provokes animmune response. This immune response can involve either antibodyproduction, or the activation of specific immunologically-competentcells, or both. It is readily apparent that an antigen can be generatedsynthesized, produced recombinantly or can be derived from a biologicalsample. Such a biological sample can include, but is not limited to atissue sample, a tumor sample, a cell or a biological fluid such, forexample, blood, plasma or ascites fluid.

“Anti-tumor effect” as used herein, refers to a biological effect, whichcan be manifested by a decrease in tumor volume, a decrease in thenumber of tumor cells, a decrease in the number of metastases, anincrease in life expectancy, or a decrease of various physiologicalsymptoms associated with the cancerous condition. An “anti-tumor effect”can also be manifested by a decrease in recurrence or an increase in thetime before recurrence.

“Chimeric receptor” as used herein refers to a synthetically designedreceptor comprising a ligand binding domain of an antibody or otherprotein sequence that binds to a molecule associated with the disease ordisorder and is linked via a spacer domain to one or more intracellularsignaling domains of a T cell or other receptors, such as acostimulatory domain. Chimeric receptor can also be referred to asartificial T cell receptors, chimeric T cell receptors, chimericimmunoreceptors, and chimeric antigen receptors (CARs). These CARs areengineered receptors that can graft an arbitrary specificity onto animmune receptor cell. The term Chimeric antigen receptors or “CARs” arealso considered by some investigators to include the antibody orantibody fragment, the spacer, signaling domain, and transmembraneregion. However, due to the surprising effects of modifying thedifferent components or domains of the CAR described herein, such as theepitope binding region (for example, antibody fragment, scFv, or portionthereof), spacer, transmembrane domain, and/or signaling domain), thecomponents of the CAR are frequently distinguished throughout thisdisclosure in terms of independent elements. The variation of thedifferent elements of the CAR can, for example, lead to stronger bindingaffinity for a specific epitope or antigen. “Co-stimulatory domain,” asthe term is used herein refers to a signaling moiety that provides to Tcells a signal which, in addition to the primary signal provided by forinstance the CD3 zeta chain of the TCR/CD3 complex, mediates a T cellresponse, including, but not limited to, activation, proliferation,differentiation, cytokine secretion, and the like. A co-stimulatorydomain can include all or a portion of, but is not limited to, CD27,CD28, 4-1BB, OX40, CD30, CD40, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, or a ligand thatspecifically binds with CD83. In some alternatives, the co-stimulatorydomain is an intracellular signaling domain that interacts with otherintracellular mediators to mediate a cell response including activation,proliferation, differentiation and cytokine secretion, and the like.“Coding for” are used herein refers to the property of specificsequences of nucleotides in a polynucleotide, such as a gene, a cDNA, oran mRNA, to serve as templates for synthesis of other macromoleculessuch as a defined sequence of amino acids. Thus, a gene codes for aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. A“nucleic acid sequence coding for a polypeptide” includes all nucleotidesequences that are degenerate versions of each other and that code forthe same amino acid sequence.

“Conditional” or “Inducible” as used herein refer to the nucleic acidconstruct that includes a promoter that provides for gene expression inthe presence of an inducer and does not substantially provide for geneexpression in the absence of the inducer.

“Constitutive” as used herein refer to the nucleic acid construct thatincludes a promoter that is constitutive providing for expression of apolypeptide that is continuously produced.

“Specific” or “Specificity” can refer to the characteristic of a ligandfor the binding partner or alternatively, the binding partner for theligand, and can include complementary shape, charge and hydrophobicspecificity for binding. Specificity for binding can includestereospecificity, regioselectivity and chemoselectivity. In somealternatives, a method of making a nucleic acid encoding a chimericantigen receptor is provided such that a nucleic acid encoding achimeric antigen receptor is generated.

“Regulate” or “modulate” as described herein, refers to the act ofcontrolling a biological process, or to exert a modifying or controllinginfluence on a biological or cellular process or pathway. In somealternatives, a system for inducible expression of a chimeric antigenreceptor is provided, wherein the system comprises a) a first nucleicacid comprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte,a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized, a polynucleotide coding for a transmembrane domain, and d) apolynucleotide coding for an intracellular signaling domain, and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, modulation comprises modulation ofcellular differentiation and apoptosis. In some alternatives, modulationcomprises modulation of cellular proliferation by regulation of activityof proteins. In some alternatives, the proteins are cell cycleregulators and transcription factors. In some alternatives, the cellcycle regulators are cyclin D1, p21, p27 and/or cdc25A. In somealternatives, the transcription factors are c-Myc.

“Cytotoxic T lymphocyte” (CTL) as used herein refers to a T lymphocytethat expresses CD8 on the surface thereof (e.g., a CD8⁺ T cell). In somealternatives such cells are preferably “memory” T cells (T_(M) cells)that are antigen-experienced.

“Central memory” T cell (or “T_(CM)”) as used herein refers to anantigen experienced CTL that expresses CD62L, CCR-7 and/or CD45RO on thesurface thereof, and does not express or has decreased expression ofCD45RA, as compared to naive cells. In some alternatives, central memorycells are positive for expression of CD62L, CCR7, CD28, CD127, CD45RO,and/or CD95, and may have decreased expression of CD54RA, as compared tonaïve cells. “Effector memory” T cell (or “T_(EM)”) as used hereinrefers to an antigen experienced T cell that does not express or hasdecreased expression of CD62L on the surface thereof, as compared tocentral memory cells, and does not express or has a decreased expressionof CD45RA, as compared to naïve cell. In some alternatives, effectormemory cells are negative for expression of CD62L and/or CCR7, ascompared to naïve cells or central memory cells, and may have variableexpression of CD28 and/or CD45RA.

“Naïve” T cells as used herein refers to a non-antigen experienced Tlymphocyte that expresses CD62L and/or CD45RA, and does not expressCD45RO−, as compared to central or effector memory cells. In somealternatives, naïve CD8+ T lymphocytes are characterized by theexpression of phenotypic markers of naïve T cells including CD62L, CCR7,CD28, CD127, and/or CD45RA.

“Effector” “T_(E)” T cells as used herein refers to antigen experiencedcytotoxic T lymphocyte cells that do not express or have decreasedexpression of CD62L, CCR7, and/or CD28, and are positive for granzyme Band/or perforin, as compared to central memory or naïve T cells.

“Enriched” and “depleted” as used herein to describe amounts of celltypes in a mixture refers to the subjecting of the mixture of the cellsto a process or step, which results in an increase in the number of the“enriched” type and a decrease in the number of the “depleted” cells.Thus, depending upon the source of the original population of cellssubjected to the enriching process, a mixture or composition may contain60, 70, 80, 90, 95, or 99 percent or more (in number or count) of the“enriched” cells and/or 40, 30, 20, 10, 5 or 1 percent or less (innumber or count) of the “depleted” cells.

“Epitope” as used herein refers to a part of an antigen or molecule thatis recognized by the immune system including antibodies, T cells, and/orB cells. Epitopes usually have at least 7 amino acids and can be linearor conformational.

“Isolated,” when used to describe the various polypeptides disclosedherein, means polypeptide or nucleic acid that has been identified andseparated and/or recovered from a component of its natural environment.Preferably, the isolated polypeptide or nucleic acid is free ofassociation with all components with which it is naturally associated.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide or nucleic acid, and can include enzymes, hormones, andother proteinaceous or non-proteinaceous solutes.

“Intracellular signaling domain” as used herein refers to all or aportion of one or more domains of a molecule (here the chimeric receptormolecule) that provides for activation of a lymphocyte. Intracellulardomains of such molecules mediate a signal by interacting with cellularmediators to result in proliferation, differentiation, activation andother effector functions. In some alternatives, such molecules includeall or portions of CD28, CD3, or 4-1BB, or combinations thereof.

“Ligand” as used herein refers to a substance that binds specifically toanother substance to form a complex. Examples of ligands includeepitopes on antigens, molecules that bind to receptors, substrates,inhibitors, hormones, and/or activators. “Ligand binding domain” as usedherein refers to substance or portion of a substance that binds to aligand. Examples of ligand binding domains include antigen bindingportions of antibodies, extracellular domains of receptors, and/oractive sites of enzymes. “Operably linked” as used herein refers tofunctional linkage between a regulatory sequence and a heterologousnucleic acid sequence resulting in expression of the latter. Forexample, a first nucleic acid sequence is operably linked with a secondnucleic acid sequence when the first nucleic acid sequence is placed ina functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Generally, operably linked DNA sequences are contiguous and, wherenecessary to join two protein coding regions, in the same reading frame.

“Percent (%) amino acid sequence identity” with respect to the chimericreceptor polypeptide sequences identified herein is defined as thepercentage of amino acid residues in a candidate sequence that areidentical with the amino acid residues in the reference sequence foreach of the ligand binding domain, spacer, transmembrane domain, and/orthe lymphocyte activating domain, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign(DNASTAR) software. Those skilled in the art can determine appropriateparameters for measuring alignment, including any algorithms needed toachieve maximal alignment over the full-length of the sequences beingcompared. For example, % amino acid sequence identity values generatedusing the WU-BLAST-2 computer program [Altschul et al., Methods inEnzymology, 266:460-480 (1996)] uses several search parameters, most ofwhich are set to the default values. Those that are not set to defaultvalues (i.e., the adjustable parameters) are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11and scoring matrix=BLOSUM62. A % amino acid sequence identity value isdetermined by dividing (a) the number of matching identical amino acidresidues between the each or all of the polypeptide amino acid sequenceof the reference chimeric receptor sequence provided in Table 2 and thecomparison amino acid sequence of interest as determined by WU-BLAST-2by (b) the total number of amino acid residues of the polypeptide ofinterest. In some alternatives, the percent sequence identity of aminoacids or nucleic acids are determined by computer software.

“Chimeric receptor variant polynucleotide” or “chimeric receptor variantnucleic acid sequence” as used herein refers to a polypeptide-encodingnucleic acid molecule as defined below having at least 80%, 85%, 90%, or95% nucleic acid sequence identity (or a percentage nucleic acidsequence identity within a range defined by any two of theaforementioned percentages) with the polynucleotide acid sequence shownin Table 1 or a specifically derived fragment thereof, such aspolynucleotide coding for an antigen binding domain, a polynucleotideencoding a spacer domain, a polynucleotide coding for a transmembranedomain and/or a polynucleotide coding for a lymphocyte stimulatorydomain. Ordinarily, a chimeric receptor variant of polynucleotide orfragment thereof will have at least 80% nucleic acid sequence identity,more preferably at least 81% nucleic acid sequence identity, morepreferably at least 82% nucleic acid sequence identity, more preferablyat least 83% nucleic acid sequence identity, more preferably at least84% nucleic acid sequence identity, more preferably at least 85% nucleicacid sequence identity, more preferably at least 86% nucleic acidsequence identity, more preferably at least 87% nucleic acid sequenceidentity, more preferably at least 88% nucleic acid sequence identity,more preferably at least 89% nucleic acid sequence identity, morepreferably at least 90% nucleic acid sequence identity, more preferablyat least 91% nucleic acid sequence identity, more preferably at least92% nucleic acid sequence identity, more preferably at least 93% nucleicacid sequence identity, more preferably at least 94% nucleic acidsequence identity, more preferably at least 95% nucleic acid sequenceidentity, more preferably at least 96% nucleic acid sequence identity,more preferably at least 97% nucleic acid sequence identity, morepreferably at least 98% nucleic acid sequence identity and yet morepreferably at least 99% nucleic acid sequence identity with the nucleicacid sequence as shown in Table or a derived fragment thereof. Variantsdo not encompass the native nucleotide sequence. In this regard, due tothe degeneracy of the genetic code, one of ordinary skill in the artwill immediately recognize that a large number of chimeric receptorvariant polynucleotides having at least 80% nucleic acid sequenceidentity to the nucleotide sequence of Table 1 will encode a polypeptidehaving an amino acid sequence which is identical to the amino acidsequence of Table 2.

“Substantially purified” refers to a molecule that has 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, or 1% or less other molecule types or other celltypes. A substantially purified cell also refers to a cell, which hasbeen separated from other cell types with which it is normallyassociated in its naturally occurring state. In some instances, apopulation of substantially purified cells refers to a homogenouspopulation of cells.

“Not substantially found” when used in reference the presence of a tumorantigen or other molecules on normal cells refers to the percentage of anormal cell type that has the antigen or molecule, and/or the density ofthe antigen on the cells. In some alternatives, not substantially foundmeans that the antigen or molecule is found on less than 50% of normalcell type and/or at a 50% less density as compared to the amount ofcells or antigen found on a tumor cell or other diseased cell.

“T cells” or “T lymphocytes” as used herein can be from any mammalian,preferably primate, species, including monkeys, dogs, and humans. Insome alternatives the T cells are allogeneic (from the same species butdifferent donor) as the recipient subject; in some alternatives the Tcells are autologous (the donor and the recipient are the same); in somealternatives the T cells arc syngeneic (the donor and the recipients aredifferent but are identical twins).

“Vector” or “construct” is a nucleic acid used to introduce heterologousnucleic acids into a cell that has regulatory elements to provideexpression of the heterologous nucleic acids in the cell. Vectorsinclude but are not limited to plasmid, minicircles, yeast, and viralgenomes. In some alternatives, the vectors are plasmid, minicircles,yeast, or viral genomes.

“Apoptosis” as described herein, refers to the process of programmedcell death (PCD) that can occur in multicellular organisms. Biochemicalevents lead to characteristic cell changes (morphology) and death. Thesechanges include blebbing, cell shrinkage, nuclear fragmentation,chromatin condensation, and chromosomal DNA fragmentation. In apoptosis,a cell initiates intracellular apoptotic signaling in response to astress, which can bring about cell suicide. The binding of nuclearreceptors by glucocorticoids, heat, radiation, nutrient deprivation,viral infection, hypoxia and increased intracellular calciumconcentration, for example, by damage to the membrane, can all triggerthe release of intracellular apoptotic signals by a damaged cell. Anumber of cellular components, such as poly ADP ribose polymerase, canalso help regulate apoptosis.

Before the actual process of cell death is precipitated by enzymes,apoptotic signals must cause regulatory proteins to initiate theapoptosis pathway. This step allows apoptotic signals to cause celldeath, or the process to be stopped, should the cell no longer need todie. Several proteins are involved, but two main methods of regulationhave been identified: targeting mitochondria functionality, or directlytransducing the signal via adaptor proteins to the apoptotic mechanisms.Another extrinsic pathway for initiation identified in several toxinstudies is an increase in calcium concentration within a cell caused bydrug activity, which also can cause apoptosis via a calcium bindingprotease calpain.

Apoptosis can be regulated by many factors. These factors can includebut are not limited to genes that can express IL-2, IL-15, Chemokinereceptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28chimeras. IL-15 regulates T and natural killer cell activation andproliferation. In rodent lymphocytes, IL15 was shown to preventapoptosis by inducing an apoptosis inhibitor, BCL2L1/BCL-X(L). In humanswith celiac disease, IL-15 similarly suppresses apoptosis inT-lymphocytes by inducing Bcl-2 and/or BCL-xL. Bcl-2 (B-cell lymphoma2), encoded in humans by the BCL2 gene, is the founding member of theBcl-2 family of regulator proteins that regulate cell death (apoptosis),by either inducing (pro-apoptotic) it or inhibiting it (anti-apoptotic).Bcl-2 is specifically considered as an important anti-apoptotic proteinand is, thus classified as an oncogene. Protein kinase B (PKB), alsoknown as Akt, is a serine/threonine-specific protein kinase that plays akey role in multiple cellular processes such as glucose metabolism,apoptosis, cell proliferation, transcription and cell migration. In somealternatives, a system for inducible expression of chimeric antigenreceptor is provided, wherein the system comprises a) a first nucleicacid comprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for acytokine, a chemokine receptor, a polypeptide that regulates apoptosis,or a polypeptide that modulates checkpoint signaling and b) a secondnucleic acid comprising a second constitutive or inducible promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the polypeptide thatregulates apoptosis or modulates checkpoint signaling comprises IL-2,IL-15, Chemokine receptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2 orPD-1CD28 chimeras.

“Checkpoint signaling” as described herein, blocks the cell cycle atspecific transition points, checkpoints to ensure that the events of thecell cycle take please in the correct order. Checkpoint signaling canalso be activated. By way of example and not of limitation, checkpointsignaling can occur by damage to the DNA so that the cell cycle does nothave to proceed until the damage is repaired. “Cell cycle checkpoints”are control mechanisms in eukaryotic cells which ensure proper divisionof the cell. Each checkpoint serves as a potential halting point alongthe cell cycle, during which the conditions of the cell are assessed,with progression through the various phases of the cell cycle occurringwhen favorable conditions are met. Currently, there are three knowncheckpoints: the G1 checkpoint, also known as the restriction or startcheckpoint; the G2/M checkpoint; and the metaphase checkpoint, alsoknown as the spindle checkpoint. The biochemical pathways that restraincell cycle transition and/or induce cell death after stress are known ascell cycle checkpoints. These checkpoints maintain the fidelity of DNAreplication, repair, and division. Polypeptides that can regulatecheckpoint signaling can include but are not limited to p53, p107, p130,and transcriptional repressor Rb.

“Negative checkpoint regulators” as described herein, refers to factorsthat can restrict the ability of T-cell responses to effectively attacktumors. They are also referred to as negative checkpoint signaling. Insome alternatives, a system for inducible expression of chimeric antigenreceptor is provided, wherein the system comprises a) a first nucleicacid comprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for acytokine, a chemokine receptor, a polypeptide that inhibits regulatesapoptosis, or a polypeptide that modulates checkpoint signaling and b) asecond nucleic acid comprising a second constitutive or induciblepromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, thepolypeptide that modulates checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3.

In another example, cell cycle inhibitors mediating the growthinhibitory cues of upstream signaling pathways, the cyclin-CDKinhibitors of the Cip/Kip family p21Cip1, p27Kip1, and p57Kip2 haveemerged as multifaceted proteins with functions beyond cell cycleregulation. In addition to regulating the cell cycle, Cip/Kip proteinscan also play important roles in apoptosis, transcriptional regulation,cell fate determination, cell migration and cytoskeletal dynamics. Acomplex phosphorylation network modulates Cip/Kip protein functions byaltering their subcellular localization, protein-protein interactions,and stability. These functions are essential for the maintenance ofnormal cell and tissue homeostasis, in processes ranging from embryonicdevelopment to tumor suppression. In some alternatives, a system forinducible expression of chimeric antigen receptor is provided, whereinthe system comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling and b) a second nucleic acid comprising a secondconstitutive or inducible promoter operably linked to nucleic acidcoding for a transcriptional activator for the inducible promoter. Insome alternatives, the polypeptide modulates checkpoint signaling. Insome alternatives, the polypeptide that modulates checkpoint signalinginhibits negative checkpoint regulators. In some alternatives, thenegative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3.

“T cell precursors” as described herein refers to lymphoid precursorcells that can migrate to the thymus and become T cell precursors, whichdo not express a T cell receptor. All T cells originate fromhematopoietic stem cells in the bone marrow. Hematopoietic progenitors(lymphoid progenitor cells) from hematopoietic stem cells populate thethymus and expand by cell division to generate a large population ofimmature thymocytes. The earliest thymocytes express neither CD4 norCD8, and are therefore classed as double-negative (CD4⁻CD8⁻) cells. Asthey progress through their development, they become double-positivethymocytes (CD4⁺CD8⁺), and finally mature to single-positive (CD4⁺CD8⁻or CD4⁻CD8⁺) thymocytes that are then released from the thymus toperipheral tissues.

About 98% of thymocytes die during the development processes in thethymus by failing either positive selection or negative selection,whereas the other 2% survive and leave the thymus to become matureimmunocompetent T cells.

The double negative (DN) stage of the precursor T cell is focused onproducing a functional β-chain whereas the double positive (DP) stage isfocused on producing a functional α-chain, ultimately producing afunctional αβ T cell receptor. As the developing thymocyte progressesthrough the four DN stages (DN1, DN2, DN3, and DN4), the T cellexpresses an invariant α-chain but rearranges the β-chain locus. If therearranged β-chain successfully pairs with the invariant α-chain,signals are produced which cease rearrangement of the β-chain (andsilence the alternate allele) and result in proliferation of the cell.Although these signals require this pre-TCR at the cell surface, theyare dependent on ligand binding to the pre-TCR. These thymocytes willthen express both CD4 and CD8 and progresses to the double positive (DP)stage where selection of the α-chain takes place. If a rearrangedβ-chain does not lead to any signaling (e.g. as a result of an inabilityto pair with the invariant α-chain), the cell may die by neglect (lackof signaling).

“Hematopoietic stem cells” or “HSC” as described herein, are precursorcells that can give rise to myeloid cells such as, for example,macrophages, monocytes, macrophages, neutrophils, basophils,eosinophils, erythrocytes, megakaryocytes/platelets, dendritic cells andlymphoid lineages (such as, for example, T-cells, B-cells, NK-cells).HSCs have a heterogeneous population in which three classes of stemcells exist, which are distinguished by their ratio of lymphoid tomyeloid progeny in the blood (L/M).

This disclosure provides for a system that has an inducible componentfor expression of transgenes and a constitutive component for expressionof transgenes. The system can be tailored to provide for regulatedexpression of one or more transgenes to provide for functionalcharacteristics in the transduced cells.

In some alternatives, a transgene under the control of the induciblepromoter is a chimeric antigen receptor (CAR). The inducible promoterprovides for the capacity to terminate CAR expression in cells whileproviding for reactivation of the cells at a later date (e.g. in thecase of relapse). In addition, the cycling of CAR T cells through on andoff periods can minimize exhaustion and/or anergy due to chronicstimulation of the T cell receptors.

The design of the vectors also provides for additional transgenes thatcan enhance one or more functional characteristics of transduced cells,such as enhanced tumor potency, survival and proliferation of transducedcells. In some alternatives, these transgenes are under the control ofan inducible promoter. Such transgenes include, without limitation,genes that promote survival and proliferation, genes that preventapoptosis, and genes that that regulate checkpoint signaling. Such genesinclude genes encoding IL-2, IL-15, Chemokine receptors, Bcl2, CA-Akt,dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras. In somealternatives, the transgenes are genes encoding IL-2, IL-15, Chemokinereceptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28chimeras. In some alternatives, the gene that modulates checkpointsignaling, encodes a polypeptide that inhibits negative checkpointregulators. In some alternatives, the negative checkpoint regulatorcomprises VISTA, LAG-3 and/or TIM3.

The disclosure provides for a system comprising first and second nucleicacids, and vectors and host cells including such nucleic acids. Each ofthe first and second nucleic acids comprise a number of modularcomponents that can be excised and replaced with other components inorder to customize the system for a specific target cell. In somealternatives, the first nucleic acid includes an inducible promoter forcontrol of the expression of the genes (e.g polynucleotide coding for achimeric antigen receptor) in an on and off manner as needed. In otheralternatives, the second nucleic acid comprises a constitutive promoterthat provides for expression of a transcriptional activator. In somealternatives, the gene encodes for a chimeric antigen receptor.

Inducible System.

The disclosure provides a system useful for providing regulatedexpression of transgenes in cells. Such transgenes include, withoutlimitation, T cell receptors, affinity matured T cell receptors,chimeric antigen receptors, chemokine receptors, cytokines, genes thatinhibit apoptosis, and/or genes that modulate checkpoint signaling. Insome alternatives, the polypeptide that modulates checkpoint signalinginhibits negative checkpoint regulators. In some alternatives, thenegative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3. Insome alternatives, the system contains a number of modular componentsthat provide for easy substitution of elements of the nucleic acid. Insome alternatives of the system, the system provides regulation ofexpression of transgenes in cells. In some alternatives, the transgenescode for T cell receptors, affinity matured T cell receptors, chimericantigen receptors, chemokine receptors, cytokines, genes that regulateapoptosis, and/or genes that modulate checkpoint signaling. In somealternatives, the gene that modulates checkpoint signaling encodes apolypeptide inhibits negative checkpoint regulators. In somealternatives, the negative checkpoint regulator comprises VISTA, LAG-3and/or TIM3.

In some alternatives, a system for inducible expression of chimericantigen receptor comprises: a first nucleic acid comprising a firstpromoter inducible by a drug, wherein the first nucleic acid is operablylinked to a polynucleotide coding for a chimeric antigen receptor, thechimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain; and a second nucleic acid comprising a second promoter operablylinked to nucleic acid coding for a transcriptional modulator for theinducible promoter. In some alternatives, the second promoter isconstitutive or inducible.

In some alternatives, a polynucleotide coding for a chimeric antigenreceptor comprises a polynucleotide coding for a ligand binding domain,wherein the target molecule is a tumor specific antigen, apolynucleotide coding for a polypeptide spacer wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and apolynucleotide coding for an intracellular signaling domain. In somealternatives, an expression vector comprises a first and/or secondnucleic acid, as described herein. Polypeptides encoded by all of or aportion of the chimeric receptor nucleic acids are also included herein.

In other alternatives, a first nucleic acid comprises a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a gene that promotes cell survival andproliferation, a gene that regulates apoptosis, and/or a gene thatmodulates checkpoint signaling. Such genes include genes encoding IL-2,IL-15, Chemokine receptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2,and/or PD-1CD28 chimeras. In some alternatives, the gene that modulatescheckpoint signaling encodes a polypeptide that inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3.

Inducible Promoters.

A system comprises a first nucleic acid comprising a first promoterinducible by a drug. By utilizing an inducible promoter, transgeneexpression can be turned on and off in order to avoid toxic side effectsand/or to allow the cells to rest during remission. Although severalinducible promoter systems are known, clinical applicability of thesesystems is limited due to toxic off target effects, unfavorablebiodistribution and pharmacodynamics profiles, limited output dynamicrange, and/or limited availability as FDA-approved commerciallyavailable pharmaceuticals. Furthermore, many of these systems usechimeric transcriptional regulators built from xenogeneic components,thus introducing the complication of immunogenicity when applying thesesystems to human therapeutics.

In some alternatives, a first promoter is inducible by a drug. The drugis selected based on safety record, favorable pharmacokinetic profile,tissue distribution, a low partition coefficient between theextracellular space and cytosol, low immunogenicity, low toxicities,and/or high expression in lymphocytes. In a specific alternative, a drugis selected that is FDA approved, provides for transgene expression inlymphocytes, does not activate other undesirable gene expression, andinduces a promoter that does not contain any xenogeneic components. Insome alternatives, the inducible promoter is activated by atranscriptional activator that interacts with a drug. Thetranscriptional activator is activated or able to bind to and activatethe inducible promoter in the presence of the drug.

A specific alternative of a drug is a drug that binds to an estrogenreceptor ligand binding domain of a transcriptional activator. In somealternatives, the drug includes tamoxifen, its metabolites, analogs, andpharmaceutically acceptable salts and/or hydrates or solvates thereof.

Tamoxifen, CAS RN: 10540-29-1, is also known as2-(4-((1Z)-1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethyl-ethanamine, or(Z)-2-(para-(1,2-Diphenyl-1-butenyl)phenoxy)-N,N-dimethylamine (IUPAC),and has a molecular formula of C₂₆H₂₉NO, M.W. 371.52. Tamoxifen is aSelective Estrogen Receptor Modulator with tissue-specific activities.Tamoxifen acts as an anti-estrogen (inhibiting agent) agent in themammary tissue, but as an estrogen (stimulating agent) in cholesterolmetabolism, bone density, and cell proliferation in the endometrium.Tamoxifen is frequently administered orally as a pharmaceuticallyacceptable salt. For example, Tamoxifen citrate (RN 54965-24-1, M.W.563.643) is indicated for treatment of metastatic breast cancer, and asan adjuvant for the treatment of breast cancer in women followingmastectomy axillary dissection, and breast irradiation. Tamoxifencitrate is also indicated to reduce incidence of breast cancer in womenat high risk for breast cancer.

Metabolites of tamoxifen in rat, mouse and human breast cancer patients,including major metabolites N-desmethyltamoxifen (RN 31750-48-8, M.W.357.494) and 4-hydroxytamoxifen (4-OHT) (RN 68392-35-8, M.W. 387.52,Afimoxifene), are disclosed in Robinson et al., Metabolites,pharmacodynamics, and pharmacokinetics of tamoxifen in rats and micecompared to the breast cancer patient. Drug Metab Dispos January 199119:36-43, which is incorporated by reference herein in its entirety.Additional cytochrome P-450 metabolites are disclosed in Crewe et al.,2002, including cis-4-hydroxytamoxifen (RN 174592, M.W. 387.52;Afimoxifene, E-isomer), and 4′ -hydroxytamoxifen((Z)-4-(1-(4-(2-(dimethylamino)ethoxy)phenyl)-1-phenylbut-1-en-2-yl)phenol).See Crewe et al., 2002, Metabolism of Tamoxifen by recombinant humancytochrome P-450 enzymes: Formation of the 4-hydroxy, 4′-hydroxy andN-desmethyl metabolites and isomerization of trans-4-hydroxytamoxifen,Drug Metab Dispos, 30(8): 869-874, FIG. 1, which is incorporated hereinby reference.

Compounds with structural similarity to tamoxifen include, but are notlimited to, cis-tamoxifen (RN 13002-65-8, M.W. 371.521),4-methyltamoxifen (RN 73717-95-5, M.W. 385.548), N-desmethyltamoxifen(RN 31750-48-8, M.W. 357.494), (Z)-desethyl methyl tamoxifen (RN15917-50-7, M.W. 357.494), (E)-desethyl methyl tamoxifen (RN 31750-45-5,M.W. 357.494), trans-4-hydoxytamoxifen (RN 68047-06-3, M.W. 387.52),Afimoxifene (RN 68392-35-8, M.W. 387.52, 4-hydroxytamoxifen),Afimoxifene, E-isomer (RN 174592-47-3, M.W. 387.52), 4-chlorotamoxifen(RN 77588-46-6, M.W. 405.966), 4-fluorotamoxifen (RN 73617-96-6, M.W.389.511), Toremifene (RN 89778-26-7, M.W. 405.966), desethyl tamoxifen(RN 19957-51-8, M.W. 343.47), (E)-desethyl tamoxifen (RN 97151-10-5,M.W. 343.47), (Z)-desethyl tamoxifen (RN 97151-11-6, M.W. 343.47),Miproxifene (RN 129612-87-9, M.W. 429.6),2-(p-(beta-ethyl-alpha-phenylstyryl)phenoxy)triethylamine (RN 749-86-0,M.W. 399.575), Droloxifene (RN 82413-20-5, M.W. 387.52),4-iodo-tamoxifen (RN 116057-68-2, M.W. 497.413), dihydrotamoxifen (RN109640-20-2, M.W. 373.537),(E)-N,N-dimethyl-2-(4-(1-(2-methylphenyl)-2-phenyl-1-butenyl)phenoxy)ethanamine(RN 97150-96-4, M.W. 385.548), or 4-hydroxytoremifene (RN 110503-62-3,M.W. 421.965); and/or pharmaceutically acceptable salts and/or hydratesor solvates thereof.

For example, citrate salts of tamoxifen, or citrate salts of compoundswith structural similarity to tamoxifen, include, but are not limited totamoxifen citrate (RN 54965-24-1, M.W. 563.64),2-(p-(1,2-diphenyl-1-butenyl)phenoxy)-N,N-dimethylethylamine citrate (RN7244-97-5, 563.64), (E)-tamoxifen citrate (RN 76487-65-5, M.W. 563.64),Toremifene citrate (RN 89778-27-8, M.W. 598.088), Droloxifene citrate(RN 97752-20-0, M.W. 579.64),2-(p-(1,2-bis(p-methoxyphenyl)-1-butenyl)phenoxy)triethylamine citrate(RN 42920-39-8, M.W. 651.748),2-(4-(1,2-diphenylethenyl)phenoxy)-N,N-diethyl-ethanamine2-hydroxy-1,2,3-propanetricarboxylate (RN 40297-42-5, M.W. 563.643),2-(p-(alpha-phenylstyryl)phenoxy)triethylamine citrate (RN 102433-95-4,M.W. 563.64),2-(p-(2-(p-methoxyphenyl)-1-phenyl-1-butenyllphenoxyltriethylaminecitrate (1:1) (RN 42824-34-0, M.W. 637.72),2-(p-(1-(p-methoxyphenyl)-2-phenylpropenyl)phenoxy)triethylamine citrate(RN 13554-24-0, M.W. 607.696),2-(p-(alpha-(p-methoxyphenyl)styryl)phenoxy)triethylamine citratemonohydrate (RN 13542-71-7, M.W. 593.669),2-(p-(p-methoxy-alpha-phenylphenethyl) phenoxy)triethylamine citrate (RN16421-72-0, M.W. 595.685),alpha-(p-(2-(diethylamino)ethoxy)phenyl)-beta-ethyl-p-methoxy-alpha-phenylphenethylalcohol citrate (1:1) (RN 35263-93-5, M.W. 639.737),1-(p-(2-(diethylamino)ethoxy)phenyl)-2-(p-methoxyphenyl)-1-phenylethanolcitrate (M.W. 611.68),alpha-p-(2-(diethylamino)ethoxy)phenyl)-beta-ethyl-alpha-(p-hydroxyphenyl)-p-methoxyphenethylalcohol citrate (RN 35263-96-8, M.W. 655.737), and/or2-(p-(p-methoxy-alpha-methylphenethyl)phenoxy)-triethylamine citrate (RN15624-34-7, M.W. 533.614).

In some alternatives, an affective amount of the drug for inducingexpression is an amount that provides for an increase in transgeneexpression over uninduced and/or basal level of expression. In somealternatives, this amount can be readily determined using known dosagesand pharmacokinetic profile of the drug.

In some alternatives, the inducible promoter has a low level of basalactivity. When a lentiviral vector is used, the level of basal activityin uninduced cells is 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% or less, ascompared to when cells are induced to express the gene. The level ofbasal activity can be determined by measuring the amount of theexpression of the transgene (e.g. marker gene) in the absence of theinducer (e.g. drug) using flow cytometry.

In some alternatives, the inducible promoter provides for a high levelof induced activity, as compared to uninduced or basal activity. In somealternatives, the level of activity in the induced state is 2, 4, 6, 8,or 10 fold or greater than the activity level in the uninduced state. Insome alternatives, transgene expression under control of the induciblepromoter is turned off in the absence of a transactivator in less than10, 8, 6, 4, 2, or 1 days excluding 0 days.

In some alternatives, an inducible promoter can be designed and/ormodified to provide for a low level of basal activity, a high level ofinducibility, and/or a short time for reversibility. In somealternatives, the inducible promoter is the 7xHBD/mE1b promoter. Anexemplary sequence for the promoter is found in Table 12 (SEQ ID NO: 41)For example, in the 7xHBD/mE1b promoter, mutations can be made toenhance the binding of the transcriptional activator.

In some alternatives, the system employs a synthetic transcriptionalactivator which, in the presence of the drug (e.g.tamoxifen), binds asynthetic promoter upstream of a transgene to induce expression. In somealternatives, the transcriptional activator is TamR-tf (HEA3). Thetamoxifen regulated transcription factor (“TamR-tf”, also designated“HEA3”) is a chimeric transcription factor composed of human subunitsincluding the N-terminal DNA binding domain of Hepatocyte Nuclear Factor1-alpha (HNF-1α) (e.g. amino acids 1-281 of SEQ ID NO: 40) fused inframe to the mutant tamoxifen-specific ligand binding domain of theestrogen receptor ligand binding domain (ER-LBD), that is in turn fusedto the p65 activation domain of NF-κB (p65). An exemplary amino acidsequence is provided in FIGS. 9A, 9B, and 9C and is identified as SEQ IDNO: 40. The mutant tamoxifen-specific ligand binding domain of theestrogen receptor ligand binding domain (ER-LBD) is found at amino acids282-595 of the TamR-tf and has a mutation at position 521. The p65activation domain of NF-κB (p65 or TAD) is found at amino acids 596 to862.

In some alternatives, a system for inducible expression of a chimericantigen receptor is provided, wherein the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora chimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the second promoter is constitutiveor inducible. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, asystem for inducible expression of chimeric antigen receptor isprovided, wherein the system comprises a) a first nucleic acidcomprising a first promoter inducible by a drug operably linked to apolynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling; and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, thepolypeptide that modulates checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3. In some alternatives, thesecond promoter is constitutive or inducible. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding forchimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment, preferably a binding fragmentthereof. In some alternatives, the ligand binding domain is single chainvariable fragment. In some alternatives, the tumor specific molecule isCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, or MAGE A3 TCR or combinations thereof. In somealternatives, the system employs a synthetic transcriptional activatorwhich, in the presence of a drug binds a synthetic promoter upstream ofa transgene to induce expression. In some alternatives, thetranscriptional activator is TamR-tf (HEA3). In some alternatives, thedrug is tamoxifen.

Additional changes can be made to the transcriptional activator toincrease the properties of the transcription factor including, withoutlimitation, altering one or more amino acids in the estrogen receptorligand binding domain and/or altering one or more amino acids in the p65transactivating domain. Altering amino acids in the estrogen receptorbinding domain can provide for more specific binding of the drug to thetranscriptional activator. An example of a transcriptional activatorwith altered amino acid sequence in the ER-LBD is shown in FIGS. 10A,10B, and 10C (SEQ ID NO:43). Mutations are made at amino acid position400, 543, and 544 of SEQ ID NO: 40. The transcriptional activator withaltered sequence has increased affinity for tamoxifen or 4-OHT. Alteringamino acids in the p65 transactivating domain can provide for increasedexpression of the transgene in the absence of activation of thetransduced cells.

In the absence of tamoxifen, TamR-tf is excluded from the nucleus bybinding of cytosolic heat-shock protein 90 (HSP90) to the tamoxifenbinding active site and transgene expression is in the “OFF” state.Nanomolar concentrations of cytosolic tamoxifen actively outcompeteHSP90 for ER-LBD binding, resulting in TamR-tf translocation to thenucleus. Upon nuclear translocation, TamR-tf is readily available tobind its restricted synthetic promoter. In the presence of tamoxifen,binding of TamR-tf to 7xHBD/EF1αp promoter induces the “ON” state oftransgene expression. In some alternatives, this transcriptionalregulator can be modified to provide for varying level of control oftransgene expression. Amino acid substitutions in the LBD of TamR-tf(HEA-3) permit selective responsiveness to tamoxifen and itsmetabolites, where 4-hydroxy tamoxifen (4-OHT) is the mostpharmacologically active metabolite, in regards to TamR-tf (HEA-3)activity, while lacking interaction with endogenous estrogen.

In some alternatives, the inducible promoter functions in a lentiviralconstruct and/or in lymphocytes.

Chimeric Antigen Receptors.

A system for expression of chimeric antigen receptor comprises: a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora chimeric antigen receptor, the chimeric antigen receptor comprising aligand binding domain, wherein the ligand binding domain is specific fora ligand, wherein the ligand is a tumor specific molecule, viralmolecule, or any other molecule expressed on a target cell population,wherein the ligand can elicit recognition, modulation, inhibition,and/or elimination by a lymphocyte; a polynucleotide coding for apolypeptide spacer, wherein the spacer is optimized; a polynucleotidecoding for a transmembrane domain; and d) a polynucleotide coding for anintracellular signaling domain. In other alternatives, anotherpolynucleotide coding for a chimeric antigen receptor is under thecontrol of a constitutive promoter. In some alternatives, the drug istamoxifen.

Ligand Binding Domain.

In some alternatives, the chimeric receptor nucleic acid comprises apolynucleotide coding for a ligand binding domain. In some alternatives,the ligand binding domain specifically binds to a tumor or viralspecific antigen and said ligand binding domain may be humanized. Insome alternatives, a ligand binding domain, includes without limitation,receptors or portions thereof, small peptides, peptidomimetics,substrates, cytokines, and the like. In some alternatives, the ligandbinding domain is an antibody or fragment thereof, preferably a bindingfragment thereof, any of which may be humanized. A nucleic acid sequencecoding for an antibody or antibody fragment can readily be determined.In a specific alternative, the polynucleotide codes for a single chainFv that specifically binds CD19. In other specific alternatives, thepolynucleotide codes for a single chain Fv that specifically binds HER2,CE7, hB7H3, or EGFR and, optionally, said polynucleotide encodes ahumanized version thereof. The sequences of these antibodies and bindingdomains thereof are known to or can readily be determined by those ofskill in the art.

Tumor antigens are proteins that are produced by tumor cells that elicitan immune response. The selection of the ligand binding domain of theinvention will depend on the type of cancer to be treated, and cantarget tumor antigens or other tumor cell surface molecules. A tumorsample from a subject can be characterized for the presence of certainbiomarkers or cell surface markers. For example, breast cancer cellsfrom a subject can be positive or negative for each of Her2Neu, Estrogenreceptor, and/or the Progesterone receptor. A tumor antigen or cellsurface molecule is selected that is found on the individual subject'stumor cells. Tumor antigens and cell surface molecules are well known inthe art and include, for example, carcinoembryonic antigen (CEA),prostate specific antigen, PSMA, Her2/neu, estrogen receptor,progesterone receptor, ephrinB2, CD19, CD20, CD22, CD23, CD123, CS-1,CE7, hB7H3, ROR1, mesothelin, c-Met, GD-2, and/or MAGE A3 TCR. In somealternatives, a target molecule is a cell surface molecule that is foundon tumor cells and is not substantially found on normal tissues, orrestricted in its expression to non-vital normal tissues.

In one alternative, the target molecule on the tumor comprises one ormore epitopes associated with a malignant tumor. Malignant tumorsexpress a number of proteins that can serve as target antigens for Tcell receptor or chimeric receptor mediated recognition. Other targetmolecules belong to the group of cell transformation-related moleculessuch as the oncogene HER-2/Neu/ErbB2. In some alternatives, the tumorantigen is selectively expressed or overexpressed on the tumor cells ascompared to control cells of the same tissue type. In otheralternatives, the tumor antigen is a cell surface polypeptide.

Once a tumor cell surface molecule that might be targeted with achimeric receptor is identified, an epitope of the target molecule isselected and characterized. Antibodies that specifically bind a tumorcell surface molecule can be prepared using methods of obtainingmonoclonal antibodies, methods of phage display, methods to generatehuman or humanized antibodies, or methods using a transgenic animal orplant engineered to produce human antibodies. Phage display libraries ofpartially or fully synthetic antibodies are available and can bescreened for an antibody or fragment thereof that can bind to the targetmolecule. Phage display libraries of human antibodies are alsoavailable. In some alternatives, antibodies specifically bind to a tumorcell surface molecule and do not cross react with nonspecific componentssuch as bovine serum albumin or other unrelated antigens. Onceidentified, the amino acid sequence or polynucleotide sequence codingfor the antibody can be isolated and/or determined.

Antibodies or antigen binding fragments include all or a portion ofpolyclonal antibodies, a monoclonal antibody, a human antibody, ahumanized antibody, a synthetic antibody, a chimeric antibody, abispecific antibody, a minibody, and a linear antibody. “Antibodyfragments” comprise a portion of an intact antibody, preferably theantigen binding or variable region of the intact antibody and canreadily be prepared. Examples of antibody fragments include Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments. In some alternatives the antibody fragments are Fab, Fab′,F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chainantibody molecules; or multispecific antibodies formed from antibodyfragments. Any of such aforementioned antibodies or antibody fragmentscan be humanized and used with the compositions and methods describedherein.

In some alternatives, a number of different antibodies that bind to aparticular tumor cell surface molecules can be isolated andcharacterized. In some alternatives, the antibodies are characterizedbased on epitope specificity of the targeted molecule. In addition, insome cases, antibodies that bind to the same epitope can be selectedbased on the affinity of the antibody for that epitope. In somealternatives, an antibody has an affinity of at least 1 mM, andpreferably <50 nM. In some alternatives, the antibody has an affinity of50 nM, 100 nM, 200 nM, 300nM 400 nM, 500 nM, 1 uM, 100 uM, 200 uM, 300uM, 400 uM, 500 uM, 600 uM, 700 uM, 800 uM, 900 uM or 1 mM or anaffinity within a range defined by any two of the aforementioned values.In some alternatives, an antibody is selected that has a higher affinityfor the epitope, as compared to other antibodies. For example, anantibody is selected that has at least a 2 fold, at least a 5 fold, atleast a 10 fold, at least a 20 fold, at least a 30 fold, at least a 40fold, or at least a 50 fold greater affinity than a reference antibodythat binds to the same epitope or an affinity that is greater than areference antibody within a range defined by any two of theaforementioned values.

In some alternatives, target molecules are CD19, CD20, CD22, CD23, CE7,hB7H3, EGFR, CD123, CS-1, ROR1, mesothelin, Her2, c-Met, PSMA, GD-2, orMAGE A3 TCR or combinations thereof. In some alternatives, the antibodyor binding fragment there of specific for these target molecules ishumanized.

In specific alternatives, the target antigen is CD19. A number ofantibodies specific for CD19 are known to those of skill in the art andcan be readily characterized for sequence, epitope binding, andaffinity. In a specific alternative, the chimeric receptor constructincludes a scFV sequence from a FMC63 antibody. In other alternatives,the scFV is a human or humanized ScFv comprising a variable light chaincomprising a CDRL1 sequence of RASQDISKYLN (SEQ ID NO: 88), CDRL2sequence of SRLHSGV(SEQ ID NO: 89), and a CDRL3 sequence of GNTLPYTFG(SEQ ID NO: 90). In other alternatives, the scFV is a human or humanizedScFv comprising a variable heavy chain comprising CDRH1 sequence ofDYGVS (SEQ ID NO: 91), CDRH2 sequence of VIWGSETTYYNSALKS (SEQ ID NO:92), and a CDRH3 sequence of YAMDYWG (SEQ ID NO: 93). The disclosurealso contemplates variable regions that have at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity tothat of the scFv for FMC63 and that have at least the same affinity forCD19.

In some alternatives, CDR regions are found within antibody regions asnumbered by Kabat as follows: for the light chain; CDRL1 amino acids24-34; CDRL2 amino acids 50-56; CDRL3 at amino acids 89-97; for theheavy chain at CDRH1 at amino acids 31-35; CDRH2 at amino acids 50-65;and for CDRH3 at amino acids 95-102. CDR regions in antibodies can bereadily determined.

In specific alternatives, the target antigen is Her2. A number ofantibodies specific for Her2 are known to those of skill in the art andcan be readily characterized for sequence, epitope binding, andaffinity. In a specific alternative, the chimeric receptor constructincludes a scFV sequence from a Herceptin antibody. In otheralternatives, the scFV is a human or humanized ScFv comprising avariable light chain comprising a CDRL1 sequence, CDRL2 sequence and aCDRL3 sequence of the Herceptin antibody. In other alternatives, thescFV is a human or humanized ScFv comprising a variable heavy chaincomprising CDRH1 sequence, CDRH2, and a CDRH3 sequence of Herceptin. TheCDR sequences can readily be determined from the amino acid sequence ofHerceptin. The disclosure also contemplates variable regions that haveat least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acidsequence identity to that of the scFv for Herceptin and that have atleast the same affinity for Her2.

“Humanized antibodies,” as described herein, refers to antibodies fromnon-human species whose protein sequences have been modified to increasetheir similarity to antibody variants produced naturally in humans. Theprocess of “humanization” can be applied to monoclonal antibodiesdeveloped for administration to humans (for example, antibodiesdeveloped as anti-cancer drugs). Humanization can be desirable when theprocess of developing a specific antibody involves utilization of anon-human immune system (such as that in mice). The protein sequences ofantibodies produced in this way are partially distinct from homologousantibodies occurring naturally in humans, and are therefore potentiallyimmunogenic when administered to human patients. Humanized antibodiesare distinct from chimeric antibodies, in that they have the proteinsequences made more similar to human antibodies but can carry a largerstretch of non-human protein. A derivative of a humanized antibody canrefer to a segment of an antibody or sequence that is derived from ahumanized antibody. In some alternatives, the ligand binding domaincomprises a humanized antibody or portion thereof. In some alternatives,the ligand binding domain comprises a scFv. In some alternatives, thescFv is a humanized scFv.

Humanization can be desirable in some alternatives for reducing theimmunogenicity of monoclonal antibodies that are derived from xenogeneicsources, such as, for example, rodents. Humanization is also desirablein some alternatives so as to improve the interaction of the antibody ora fragment thereof with the human immune system. Due to the developmentof hybridoma technology, a large number of xenogeneic antibodies arehighly immunogenic in humans, which can ultimately limit their clinicalapplications especially when administration may need to be repeated.Additionally, they can be rapidly removed from the circulation and cancause systemic inflammatory effects as well. Therefore humanizationstrategies are desirable in some alternatives to circumvent thesesituations. Techniques for antibody humanization are known to thoseskilled in the art.

In some alternatives, a polynucleotide coding for a ligand bindingdomain is operably linked to a polynucleotide coding for a spacerregion. In some alternatives, the polynucleotide coding for a ligandbinding domain can also have one or more restriction enzyme sites at the5′ and/or 3′ ends of the coding sequence in order to provide for easyexcision and replacement of the polynucleotide with anotherpolynucleotide coding for a ligand binding domain coding for a differentantigen or that has different binding characteristics. For example, arestriction site, NheI, is encoded upstream of the leader sequence; anda 3′ RsrII located within the hinge region allows subcloning of anydesirable scFv into a chimeric receptor vector. In some alternatives,the polynucleotide is codon optimized for expression in mammalian cells.

In some alternatives, the polynucleotide coding for a ligand bindingdomain is operably linked to a signal peptide. In some alternatives, thesignal peptide is a signal peptide for granulocyte colony stimulatingfactor. Polynucleotides coding for other signal peptides such as CD8alpha can be utilized. In some alternatives, the polynucleotide codesfor CD8 alpha.

In some alternatives, the polynucleotide coding for a ligand bindingdomain is operably linked to a promoter. A promoter is selected thatprovides for expression of the chimeric antigen receptor in a mammaliancell. In a specific alternative, the promoter is an inducible promoter.

A specific alternative of a polynucleotide coding for a ligand bindingdomain is shown in Table 1 as the scFv from an antibody thatspecifically binds CD19, such as FMC63. A polynucleotide encoding for aflexible linker including the amino acids GSTSGSGKPGSGEGSTKG (SEQ ID NO:94) separates the VH and VL chains in the scFV. The amino acid sequenceof the scFv including the linker is shown in Table 2. (SEQ ID NO:11)Other CD19-targeting antibodies such as SJ25C1 and HD37 are known.(SJ25C1: Bejcek et al. Cancer Res 2005, PMID 7538901; HD37: Pezutto etal. JI 1987, PMID 2437199).

Spacer.

In some alternatives, the chimeric receptor nucleic acid comprises apolynucleotide coding for a spacer region. Typically a spacer region isfound between the ligand binding domain and the transmembrane domain ofthe chimeric receptor. In some alternatives, a spacer region providesfor flexibility of the ligand binding domain and allows for highexpression levels in lymphocytes. A CD19-specific chimeric receptorhaving a spacer domain of 229 amino acids had less antitumor activitythan a CD19-specific chimeric receptor with a short spacer regioncomprised of the modified IgG4 hinge only. In some alternatives, thespacer is optimized for increased T cell proliferation and/or cytokineproduction in response to the ligand as compared to a reference chimericreceptor.

In some alternatives, a spacer region has at least 10 to 229 aminoacids, 10 to 200 amino acids, 10 to 175 amino acids, 10 to 150 aminoacids, 10 to 125 amino acids, 10 to 100 amino acids, 10 to 75 aminoacids, 10 to 50 amino acids, 10 to 40 amino acids, 10 to 30 amino acids,10 to 20 amino acids, or 10 to 15 amino acids, or a length within arange defined by any two of the aforementioned lengths. In somealternatives, a spacer region has 12 amino acids or less, 119 aminoacids or less, or 229 amino acids or less but greater than 1 or 2 aminoacids. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor.

In some alternatives, the spacer region is derived from a hinge regionof an immunoglobulin like molecule. In some alternatives, a spacerregion comprises all or a portion of the hinge region from a human IgG1,human IgG2, a human IgG3, or a human IgG4, and can contain one or moreamino acid substitutions. Exemplary sequences of the hinge regions areprovided in Table 8. In some alternatives, a portion of the hinge regionincludes the upper hinge amino acids found between the variable heavychain and the core, and the core hinge amino acids including apolyproline region. In some alternatives, the spacer is optimized forincreased T cell proliferation and/or cytokine production in response tothe ligand as compared to a reference chimeric receptor.

In some alternatives, hinge region sequences can be modified in one ormore amino acids in order to avoid undesirable structural interactionssuch as dimerization. In a specific alternative, the spacer regioncomprises a portion of a modified human hinge region from IgG4, forexample, as shown in Table 2 or Table 8 (SEQ ID NO: 21). Arepresentative of a polynucleotide coding for a portion of a modifiedIgG4 hinge region is provided in Table 1. (SEQ ID NO: 4). In somealternatives, a hinge region can have at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity with a hinge region aminoacid sequence identified in Table 2 or Table 8, or any other percentsequence identity between any two of the percent sequence identitieslisted. In a specific alternative, a portion of a human hinge regionfrom IgG4 has an amino acid substitution in the core amino acids fromCPSP to CPPC.

In some alternatives, all or a portion of the hinge region is combinedwith one or more domains of a constant region of an immunoglobulin. Forexample, a portion of a hinge region can be combined with all or aportion of a CH2 or CH3 domain or variant thereof. In some alternatives,the spacer region does not include the 47-48 amino acid hinge regionsequence from CD8apha or the spacer region consisting of anextracellular portion of the CD28 molecule.

In some alternatives, a short spacer region has 12 amino acids or lessand comprises all or a portion of a IgG4 hinge region sequence orvariant thereof, an intermediate spacer region has 119 amino acids orless and comprises all or a portion of a IgG4 hinge region sequence anda CH3 region or variant thereof, and a long spacer has 229 amino acidsor less and comprises all or a portion of a IgG4 hinge region sequence,a CH2 region, and a CH3 region or variant thereof. In some alternatives,a short spacer region has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 aminoacids or a size within a range defined by any two of the aforementionedamino acid lengths. In some alternatives, a medium spacer region has 13,20, 30, 40, 50, 60, 70, 80, 90, 100, 110 or 119 amino acids or a sizewithin a range defined by any two of the aforementioned amino acidlengths. In some alternatives, a spacer region has 120, 130, 140, 150,160, 170, 180, 190, 200, 210 or 219 amino acids or a size within a rangedefined by any two of the aforementioned amino acid lengths.

A polynucleotide coding for a spacer region can be readily prepared bysynthetic or recombinant methods from the amino acid sequence. In somealternatives, a polynucleotide coding for a spacer region is operablylinked to a polynucleotide coding for a transmembrane region. In somealternatives, the polynucleotide coding for the spacer region can alsohave one or more restriction enzyme sites at the 5′ and/or 3′ ends ofthe coding sequence in order to provide for easy excision andreplacement of the polynucleotide with another polynucleotide coding fora different spacer region. In some alternatives, the polynucleotidecoding for the spacer region is codon optimized for expression inmammalian cells.

In an alternative, the spacer region is a hinge region sequence fromIgG1, IgG2, IgG3, or IgG4 or a portion thereof, a hinge region sequencefrom IgG1, IgG2, IgG3, or IgG4 in combination with all or a portion of aCH2 region or variant thereof, a hinge region sequence from IgG1, IgG2,IgG3, or IgG4 in combination with all or a portion of a CH3 region orvariant thereof, and a hinge region sequence from IgG1, IgG2, IgG3, orIgG4 in combination with all or a portion of a CH2 region or variantthereof, and/or a CH3 region or variant thereof. In some alternatives, ashort spacer region is a modified IgG4 hinge sequence (SEQ ID NO: 4)having 12 amino acids or less but greater than one or two amino acids,an intermediate sequence is a IgG4 hinge sequence with a CH3 sequencehaving 119 amino acids or less but greater than one or two amino acids(SEQ ID NO: 62); or a IgG4 hinge sequence with a CH2 and CH3 regionhaving 229 amino acids or less but greater than one or two amino acids(SEQ ID NO: 50).

Transmembrane Domain.

In some alternatives, the chimeric receptor nucleic acid comprises apolynucleotide coding for a transmembrane domain. The transmembranedomain provides for anchoring of the chimeric receptor in the membrane.

In an alternative, the transmembrane domain that naturally is associatedwith one of the domains in the chimeric receptor is used. In some cases,the transmembrane domain can be selected or modified by amino acidsubstitution to avoid binding of such domains to the transmembranedomains of the same or different surface membrane proteins to minimizeinteractions with other members of the receptor complex.

The transmembrane domain can be derived either from a natural or asynthetic source. When the source is natural, the domain can be derivedfrom any membrane-bound or transmembrane protein.

Transmembrane regions comprise at least the transmembrane region(s) of)the alpha, beta or zeta chain of the T-cell receptor, CD28, CD3, CD45,CD4, CD8, CD9, CD16, CD22; CD33, CD37, CD64, CD80, CD86, CD134, CD137and/or CD154. In a specific alternative, the transmembrane domaincomprises the amino acid sequence of the CD28 transmembrane domain asshown in Table 2. A representative polynucleotide sequence coding forthe CD28 transmembrane domain is shown in Table 1 (SEQ ID NO: 5).

A transmembrane domain can be synthetic or a variant of a naturallyoccurring transmembrane domain. In some alternatives, synthetic orvariant transmembrane domains comprise predominantly hydrophobicresidues such as leucine and valine. In some alternatives, atransmembrane domain can have at least 80%, 85%, 90%, 95%, or 100% aminoacid sequence identity with a transmembrane domain as shown in Table 2or Table 6 or an amino acid sequence identity within a range defined byany two of the aforementioned values. Variant transmembrane domainspreferably have a hydrophobic score of at least 50 as calculated by KyteDoolittle.

A polynucleotide coding for a transmembrane domain can be readilyprepared by synthetic or recombinant methods. In some alternatives, apolynucleotide coding for a transmembrane domain is operably linked to apolynucleotide coding for an intracellular signaling region. In somealternatives, the polynucleotide coding for a transmembrane domain canalso have one or more restriction enzyme sites at the 5′ and/or 3′ endsof the coding sequence in order to provide for easy excision andreplacement of the polynucleotide coding for a transmembrane domain withanother polynucleotide coding for a different transmembrane domain. Insome alternatives, the polynucleotide coding for a transmembrane domainis codon optimized for expression in mammalian cells. In somealternatives, the mammalian cells are human cells.

Intracellular Signaling Domain.

In some alternatives, the chimeric receptor nucleic acid comprises apolynucleotide coding for an intracellular signaling domain. Theintracellular signaling domain provides for activation of one functionof the transduced cell expressing the chimeric receptor upon binding tothe ligand expressed on tumor cells. In some alternatives, theintracellular signaling domain contains one or more intracellularsignaling domains. In some alternatives, the intracellular signalingdomain is a portion of and/or a variant of an intracellular signalingdomain that provides for activation of at least one function of thetransduced cell.

Examples of intracellular signaling domains for use in a chimericreceptor of the disclosure include the cytoplasmic sequences of the CD3zeta chain, and/or co-receptors that act in concert to initiate signaltransduction following chimeric receptor engagement, as well as anyderivative or variant of these sequences and any synthetic sequence thathas the same functional capability. T cell activation can be said to bemediated by two distinct classes of cytoplasmic signaling sequence:those that initiate antigen-dependent primary activation and provide a Tcell receptor like signal (primary cytoplasmic signaling sequences) andthose that act in an antigen-independent manner to provide a secondaryor co-stimulatory signal (secondary cytoplasmic signaling sequences).Primary cytoplasmic signaling sequences that act in a stimulatory mannercan contain signaling motifs which are known as receptor tyrosine-basedactivation motifs or ITAMs. Examples of ITAM containing primarycytoplasmic signaling sequences include those derived from CD3 zeta, FcRgamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b,and/or CD66d. In some alternatives, the primary signaling intracellulardomain can have at least 80%, 85%, 90%, or 95% sequence identity toCD3zeta having a sequence provided in Table 2 or at least a percentsequence identity that is within a range defined by any two of thepercent sequence identities listed. In some alternatives of thevariants, of CD3 zeta retain at least one, two, three or all ITAMregions as shown in Table 7.

In a preferred alternative, the intracellular signaling domain of thechimeric receptor can be designed to comprise the CD3-zeta signalingdomain by itself or combined with any other desired cytoplasmicdomain(s). For example, the intracellular signaling domain of thechimeric receptor can comprise a CD3zeta chain and a costimulatorysignaling region.

The costimulatory signaling region refers to a portion of the chimericreceptor comprising the intracellular domain of a costimulatorymolecule. A costimulatory molecule is a cell surface molecule other thanan antigen receptor or their ligands that is required for a response oflymphocytes to an antigen. Examples of such molecules include CD27,CD28, 4-1BB (CD 137), OX40, CD30, CD40, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, zeta chain associatedprotein kinase (ZAP70), and/or a ligand that specifically binds withCD83. In some alternatives, the costimulatory signaling domain can haveat least 80%, 85%, 90%, or 95% amino acid sequence identity to theintracellular domain of CD28 as shown in Table 5 or to 4-1BB having asequence provided in Table 2 or at least a percent sequence identitythat is within a range defined by any two of the percent sequenceidentities listed. In an alternative, a variant of the CD28intracellular domain comprises an amino acid substitution at positions186-187, wherein LL is substituted with GG.

The intracellular signaling sequences of the chimeric receptor can belinked to each other in a random or specified order. In somealternatives, a short oligo- or polypeptide linker, preferably between 2and 10 amino acids in length can form the linkage. In one alternative,the intracellular signaling domains comprises all or a portion of thesignaling domain of CD3-zeta or variant thereof and all or a portion ofthe signaling domain of CD28 or a variant thereof. In anotheralternative, the intracellular signaling domain comprises all or aportion of the signaling domain of CD3-zeta or variant thereof and allor a portion of the signaling domain of 4-1BB or variant thereof. In yetanother alternative, the intracellular signaling domain comprises all ora portion of the signaling domain of CD3-zeta or variant thereof, all ora portion of the signaling domain of CD28 or variant thereof, and all ora portion of the signaling domain of 4-1BB or variant thereof. In aspecific alternative, the amino acid sequence of the intracellularsignaling domain comprising a variant of CD3zeta and a portion of the4-1BB intracellular signaling domain is provided in Table 2. Arepresentative nucleic acid sequence is provided in Table 1 (SEQ ID NO:6; SEQ ID NO: 7). In some alternatives, the nucleic acid sequencecomprises the sequence set forth in SEQ ID NO: 6. In some alternatives,the nucleic acid sequence comprises the sequence set forth in SEQ ID NO:7.

In an alternative, a polynucleotide coding for an intracellularsignaling domain comprises a 4-1BB intracellular domain linked to aportion of a CD3zeta domain. In other alternatives, a 4-1BBintracellular domain and a CD28 intracellular domain are linked to aportion of a CD3 zeta domain.

A polynucleotide coding for an intracellular signaling domain can bereadily prepared by synthetic or recombinant methods from the amino acidsequence. In some alternatives, the polynucleotide coding for anintracellular signaling domain can also have one or more restrictionenzyme sites at the 5′ and/or 3′ ends of the coding sequence in order toprovide for easy excision and replacement of the polynucleotide codingfor an intracellular signaling domain with another polynucleotide codingfor a different intracellular signaling domain. In some alternatives,the polynucleotide coding for an intracellular signaling domain is codonoptimized for expression in mammalian cells. In some alternatives, themammalian cells are human cells.

Marker Sequences.

In some alternatives, the system further comprises one or more markersequences under the control of an inducible promoter. A marker sequencecan provide for selection of transduced cells, and/or identification oftransduced cells. In some alternatives, the marker sequence is for aselection of transduced cells and/or identification of transduced cells.In some alternatives, the marker sequence is operably linked to apolynucleotide sequence coding for a linker sequence. In somealternatives, the linker sequence is a cleavable linker sequence. Insome alternatives, the linker is a cleavable T2A linker.

A number of different marker sequences can be employed. Typically amarker sequence has a functional characteristic that allows forselection of transduced cells and/or detection of transduced cells. Insome alternatives, the marker sequence is compatible with transductionof human lymphocytes. In some alternatives, the marker sequence allowsfor selection of transduced cells and/or detection of transduced cells.

The positive selectable marker can be a gene, which upon beingintroduced into the host cell, expresses a dominant phenotype permittingpositive selection of cells carrying the gene. Genes of this type areknown in the art, and include, inter alia, hygromycin-Bphosphotransferase gene (hph) which confers resistance to hygromycin B,the amino glycoside phosphotransferase gene (neo or aph) from Tn5, whichcodes for resistance to the antibiotic G418, the dihydrofolate reductase(DHFR) gene, which provides resistance to methotrexate, DHFR dm(exemplary polynucleotide and amino acid sequences in FIG. 12, SEQ IDNO: 46 and SEQ ID NO: 47, the pac gene that provides resistance topuromycin, Sh ble gene which inactivates zeocin, the adenosine deaminasegene (ADA), and the multi-drug resistance (MDR) gene. Transduced cellscultured in the presence of these agents will survive and be selected.

In an alternative, a first nucleic acid further comprises apolynucleotide coding for a marker sequence. In an alternative, themarker sequence is a truncated epidermal growth factor receptor as shownin Table 2. An exemplary polynucleotide for the truncated epidermalgrowth factor receptor is shown in Table 1. (SEQ ID NO: 9). In somealternatives, the marker sequence is a truncated Her2 sequence. Anexemplary polynucleotide and amino acid for the truncated Her2 sequencesis shown in FIG. 11 and provided by SEQ ID NO: 44 and SEQ ID NO: 45,respectively.

In some alternatives, the polynucleotide coding for the marker sequenceis operably linked to a polynucleotide coding for a linker sequence. Ina specific alternative, the linker sequence is a cleavable linkersequence T2A, as shown in Table 2. An exemplary polynucleotide sequencecoding for the T2A linker is provided in Table 1. (SEQ ID NO:8).

A polynucleotide coding for marker sequence can be readily prepared bysynthetic or recombinant methods from the amino acid sequence. In somealternatives, a polynucleotide coding for a marker sequence is operablylinked to a polynucleotide coding for an intracellular signaling domain.In some alternatives, the polynucleotide coding for a marker sequencecan also have one or more restriction enzyme sites at the 5′ and/or 3′ends of the coding sequence in order to provide for easy excision andreplacement of the polynucleotide coding for a marker sequence withanother polynucleotide coding for a different marker sequence. In somealternatives, the polynucleotide coding for a marker sequence is codonoptimized for expression in mammalian cells, preferably humans.

In some alternatives, two or more marker sequences can be employed. Insome alternatives, a first marker sequence is under control of aconstitutive promoter and provides for an indication that the transducedcell is expressing the transgene. In other alternatives, a second markersequence is under the control of the inducible promoter and provides anindication that the transgene expression has been induced. In somealternatives, the marker under the control of the inducible promoter canbe used to select for cells in which noninduced or basal expression ismuch lower than in other cells by selecting cells that have a lowerexpression of the marker sequence under the control of the induciblepromoter and expand those cells for further applications.

Other Genetic Components Under Control of Inducible Promoter.

In some alternatives, the first nucleic acid comprises a polynucleotidesequence coding for genes that promote survival and proliferation, genesthat prevent apoptosis, and/or genes that inhibit negative checkpointsignaling under the control of an inducible promoter. Such genes includegenes encoding IL-2, IL-15, Chemokine receptors, Bcl2, CA-Akt,dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras. These genes arealso placed under the control of an inducible promoter as describedherein. In some alternatives, the genes encode IL-2, IL-15, Chemokinereceptors, Bcl2, CA-Akt, dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28chimeras. In some alternatives, the gene that modulates checkpointsignaling encodes a polypeptide that inhibits negative checkpointregulators. In some alternatives, the negative checkpoint regulatorcomprises VISTA, LAG-3 and/or TIM3.

In some alternatives, a first nucleic acid comprises a first induciblepromoter linked to a polynucleotide coding for a cytokine or chemokinereceptor. Chemokines, also referred to as chemotactic cytokines, are agroup of structurally related proteins that regulate cell trafficking oflymphocytes. In some alternatives, the chemokines are homeostatic orinflammatory. Chemokine receptors include CCR2, CCR7, or CCR15.Cytokines include interleukins such as IL2, IL-12, IL-7, and/or Il-15,interferons, such as interferon 6, tumor necrosis factor, and a TLR4agonist. In some alternatives, the chemokine receptors comprise CCR2,CCR7, and/or CCR15. In some alternatives, the chemokine receptorsinclude CCR2, CCR7, or CCR15. In some alternatives, the cytokinesinclude interleukins, wherein the interleukins are IL2, IL-12, IL-7and/or Il-15 or interferons, wherein the interferons comprise interferon6, tumor necrosis factor, or a TLR4 agonist.

In some alternatives, a first nucleic acid comprises a first induciblepromoter linked to a polynucleotide coding for a polypeptide thatregulates apoptosis. In some alternatives, genes that inhibit apoptosisinclude, for example, Bcl2, and/or CA-Akt. In some alternatives, thepolypeptide is Bcl2 or CA-Akt.

In some alternatives, a first nucleic acid comprises a first induciblepromoter linked to a polynucleotide coding for a polypeptide thatmodulates checkpoint signaling. Such genes include dn-TGFbetaRIII,dn-SHP1/2, and/or PD-1CD28 chimeras. In some alternatives, thepolypeptide is dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras. Insome alternatives, the polypeptide that modulates checkpoint signalinginhibits negative checkpoint regulators. In some alternatives, thenegative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3.

Exemplary sequences for polynucleotides encoding these genes are foundat:

Gene A.A. A.A. N.A. N.A. Accession gI Accession gI CCR2 P41597 1168965NM_001123041 183979979 CCR7 P32248 1352335 NM_001838 299473754 IL-2AAH70338 47682793 BC070338 47682792 IL-12 AAD16432 4323579 AF1010624323578 IL-7 AAC63047 386824 NM_000880 315467865 IL-15 CAG46804 49456967CR542007 49456966 IFN-g EAW97180 119617586 EAW97180 119617586 TNFNP_000585 25952111 NM_000594 395132451 Bcl2 AAH27258 20072668 BC02725820072667 CA-Akt NP_001014432 62241015 NM_001014432 62241014 TGFbetaReceptor III Q03167 311033535 NM_003243 307574689 SHP1 NP_00282218104989 NM_002831 166064064 SHP2 Q06124 84028248 NM_002834 33356176PD-1 NP_001129245 209413749 NM_001135773 209413748 CD28 AAA51945 180092NM_006139 340545506

Any number of nucleic acids can be placed under the control of aninducible promoter including those coding for chimeric antigen receptor,a marker sequences, a cytokine, a chemokine, an inhibitor of apoptosis,and/or an inhibitor of negative checkpoint signaling. In somealternatives, one or more inducible promoters can be utilized to providefor an adequate expression level of each of the nucleic acids. In somealternatives, constructs can be prepared with a gene such as a cytokineunder the control of an inducible promoter and a construct comprising achimeric antigen receptor under the control of a constitutive promoter.Such constructs are useful to provide for cell survival andproliferation of transduced cells, for example lymphocytes expressing achimeric antigen receptor.

Constitutive Promoter Systems.

In other alternatives, a system comprises a second nucleic acid thatcomprises a constitutive promoter or a second inducible promoter linkedto a transcriptional activator. In other alternatives, a systemcomprises a second nucleic acid that comprises a promoter linked to atranscriptional activator. In some alternatives, the promoter is aconstitutive promoter or an inducible promoter. In some alternatives, aconstitutive promoter includes EF1α promoter, actin promoter, the myosinpromoter, the hemoglobin promoter, and the creatine kinase promoter. Insome alternatives, viral promoters such as the CMV promoter areexcluded. In some alternatives, the constitutive promoter can be linkedto one or more of a polynucleotide coding for marker, or a chimericantigen receptor as described herein.

Constitutive Promoters.

A constitutive promoter provides for continuous gene expression of thegene under the control of the promoter. In some alternatives, theconstitutive promoter is a promoter that provides for gene expression ina lentiviral construct and/or in lymphocytes. In some alternatives, thepromoter is not derived from a xenogenic source such as a plant or avirus.

In a specific alternative, the constitutive promoter comprises EF1αpromoter, actin promoter, the myosin promoter, the hemoglobin promoter,and/or the creatine kinase promoter. In some alternatives, viralpromoters such as the CMV promoter are excluded.

Transcriptional Activators.

In some alternatives, the constitutive promoter is operably linked to atranscriptional activator. In some alternatives, the transcriptionalactivator activates an inducible promoter in the presence of the inducer(e.g. drug).

In some alternatives, an inducible promoter is induced in the presenceof a transcriptional activator. In some alternatives, thetranscriptional activator preferentially binds to the promoter in thepresence of the drug. In some alternatives, the transcriptionalactivator is TamR-tf (HEA3). Modification of the transcriptionalactivator can be made in the amino acid sequence that can affect theability of activator to bind to the drug, the promoter, or both. Forexample, binding in the ER ligand binding domain would affect thebinding of the drug to the transcriptional activator.

In some alternatives, the system employs a synthetic transcriptionalactivator which, in the presence of the drug (e.g. tamoxifen), binds asynthetic promoter upstream of a transgene to induce expression. In somealternatives, the transcriptional activator is TamR-tf (HEA3). Thetamoxifen regulated transcription factor (“TamR-tf”, also designated“HEA3”) is a chimeric transcription factor composed of human subunitsincluding the N-terminal DNA binding domain of Hepatocyte Nuclear Factor1-alpha (HNF-1α) (e.g. amino acids 1-281 of SEQ ID NO: 40) fused inframe to the mutant tamoxifen-specific ligand binding domain of theestrogen receptor ligand binding domain (ER-LBD), that is in turn fusedto the p65 activation domain of NF-κB (p65). An exemplary amino acidsequence is provided in FIGS. 9A, 9B and 9C and is identified as SEQ IDNO: 40. The mutant tamoxifen-specific ligand binding domain of theestrogen receptor ligand binding domain (ER-LBD) is found at amino acids282-595 of the TamR-tf and has a mutation at position 521. The p65activation domain of NF-κB (p65 or TAD) is found at amino acids 596 to862.

Additional changes can be made to the transcriptional activator toincrease the properties of the transcription factor including, withoutlimitation, altering one or more amino acids in the estrogen receptorligand binding domain and/or altering one or more amino acids in the p65transactivating domain. Altering amino acids in the estrogen receptorbinding domain can provide for more specific binding of the drug to thetranscriptional activator. An example of a transcriptional activatorwith altered sequence in the ER-LBD is shown in FIGS. 10A, 10B and 10C(SEQ ID NO:43). Mutations are made at amino acid position 400, 543, and544 of SEQ ID NO: 40. The transcriptional activator with alteredsequence has increased affinity for tamoxifen or 4-OHT. Altering aminoacids in the p65 transactivating domain can provide for increasedexpression of the transgene in the absence of activation of thetransduced cells.

Marker.

In some alternatives, the constitutive promoter is operably linked to apolynucleotide coding for a marker polypeptide. Such marker polypeptidesare described herein, and include EGFRt, Her2t, and/or DHFRdm.

Chimeric Antigen Receptor.

In some alternatives, the constitutive promoter is operably linked to apolynucleotide coding for a chimeric antigen receptor. In somealternatives, the chimeric antigen receptor comprises a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized to provide forincreased T cell proliferation and/or cytokine production in response tothe ligand as compared to a reference chimeric receptor. Examples ofchimeric antigen receptors are described herein.

Vectors.

A variety of vector combinations can be constructed to provide forefficiency of transduction and transgene expression. In somealternatives, the vector is a dual packaged or single (all in one) viralvector. In other alternatives, the vectors can include a combination ofviral vectors and plasmid vectors. Other viral vectors include foamyvirus, adenoviral vectors, retroviral vectors, and lentiviral vectors.In some alternatives, the vector is a lentiviral vector. In somealternatives, the vector is a foamy viral vector, adenoviral vectors,retroviral vectors or lentiviral vectors.

In some alternatives, a plasmid vector or a viral vector comprises afirst nucleic acid comprising an inducible promoter linked to apolynucleotide coding for a chimeric antigen receptor. In somealternatives, a plasmid vector or viral vector comprises a first nucleicacid sequence comprising a polynucleotide coding for a gene thatenhances cell survival or proliferation, a gene that regulatesapoptosis, and/or a gene that modulates checkpoint signaling. In somealternatives, the modulation of checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3. Such polynucleotides codefor a cytokine, or a chemokine receptor. In some alternatives, a plasmidvector or a viral vector comprises a first nucleic acid comprising aninducible promoter linked to a polynucleotide coding for a markersequence. Marker sequences are described herein. In some alternatives,the marker sequence is compatible with transduction of humanlymphocytes. In some alternatives, the marker sequence allows forselection of transduced cells and/or detection of transduced cells. Insome alternatives, the marker is a gene that can include inter alia,hygromycin-B phosphotransferase gene (hph), which confers resistance tohygromycin B, the amino glycoside phosphotransferase gene (neo or aph)from Tn5, which codes for resistance to the antibiotic G418, thedihydrofolate reductase (DHFR) gene, which provides resistance tomethotrexate, DHFR dm (exemplary polynucleotide and amino acid sequencesin FIG. 12, SEQ ID NO:46 and SEQ ID NO:47, the pac gene that providesresistance to puromycin, Sh ble gene, which inactivates zeocin, theadenosine deaminase gene (ADA), and/or the multi-drug resistance (MDR)gene. A first nucleic acid can include a number of differentpolynucleotide sequences all under the control of the induciblepromoter. For example, a polynucleotide coding for a chimeric antigenicreceptor can be linked to a polynucleotide coding for a markerpolypeptide and/or a polynucleotide coding for cytokine or chemokinereceptor.

In some alternatives, a lentiviral vector comprises a second nucleicacid comprising a constitutive promoter linked to a nucleic acidsequence coding for transcriptional activator that binds to drug andactivates expression of an inducible promoter. In some alternatives, alentiviral vector with a constitutive promoter can also include anucleic acid sequence including a marker gene, piggyback transposase,and/or a polynucleotide coding for a chimeric antigen receptor. Eachelement of the nucleic acid can be separated from one another with asequence such as a T2A self-cleaving sequence. In some alternatives, theelements of the nucleic acid are separated from one another with asequence self-cleaving sequence. In some alternatives, the self-cleavingsequence is T2A.

In other alternatives, the heterogeneous (heterogeneous to the vector,e,g, lentiviral vector) nucleic acid sequence is limited by the amountof additional genetic components that can be packaged in the vector. Insome alternatives, a construct contains at least two genes heterogenousto the viral vector. In some alternatives, the construct contains nomore than 4 genes heterogenous to the viral vector. The number of genesheterogenous to the viral vector that can be packaged in the vector canbe determined by detecting the expression of one or more transgenes, andselecting vector constructs that provide for transduction of at least10% of the cells and/or detectable expression levels of the transgene inat least 10% of the cells.

In some alternatives, a lentivirus is a dual packaged virus. A dualpackaged virus contains at least one conditional construct comprising aninducible promoter operably linked to a polynucleotide coding for achimeric antigen receptor. Optionally the conditional constructcomprises a marker gene, a nucleic acid for a cytokine, a nucleic acidfor a chemokine receptor. In some alternatives, a dual packagedlentivirus contains a constitutive construct comprising a constitutivepromoter. In an alternative, the constitutive construct comprises aconstitutive promoter linked to a transcriptional activator for theinducible promoter. In some alternatives, the constitutive constructalso includes a marker gene and/or a polynucleotide encoding a cytokineor chemokine. In some alternatives of a system with two constructs, eachconstruct can be packaged in a separate viral vector and the viralvectors can be mixed together for transduction in a cell population.

When the constitutive and conditional constructs both contain a markergene, the marker gene on each construct is the same or different fromone another. In some alternatives, when the constitutive and conditionalconstructs both contain a polynucleotide coding for a chimeric antigenreceptor, the chimeric antigen receptor can be targeted to the sameantigen but have different ligand binding domains, can be targeted tothe same antigen but different epitopes, or can be targeted to differentantigens.

In some alternatives, the vector is a minicircle. Minicircles areepisomal DNA vectors that are produced as circular expression cassettesdevoid of any bacterial plasmid DNA backbone. Their smaller molecularsize enables more efficient transfections and offers sustainedexpression over a period of weeks as compared to standard plasmidvectors that only work for a few days. In some alternatives, aminicircle contains a drug inducible promoter linked to a polynucleotidecoding for a chimeric antigen receptor. In some alternatives, theinducible promoter can be linked to chemokine receptor, a marker gene,and/or a cytokine. One or more minicircles can be employed. In somealternatives, a minicircle comprises an inducible promoter linked to apolynucleotide coding for a first chimeric antigen receptor, anotherminicircle comprises an inducible promoter linked to a polynucleotidecoding for a second and different chimeric antigen receptor, and/or aminicircle comprises an inducible promoter linked to a polynucleotidecoding for a chemokine receptor, a chimeric antigen receptor, and amarker gene. Each element of the constructs is separated by a nucleicacid, such as that coding for a self-cleaving T2A sequence. In somealternatives, each element of the constructs is separated by a nucleicacid, such as that coding for a self-cleaving T2A sequence. In somealternatives each minicircle differs from one another in the chimericantigen receptor including but not limited to the spacer length andsequence, the intracellular signaling domain, and/or the markersequence. The minicircle vector can be used with a constitutivelentivirus vector coding for a transcriptional activator for theinducible promoter. In some alternatives, the minicircle vector is usedwith a constitutive lentivirus vector coding for a transcriptionalactivator for the inducible promoter.

In some alternatives, the vector is a piggy bac transposon. The PiggyBac(PB) transposon is a mobile genetic element that efficiently transposesbetween vectors and chromosomes via a “cut and paste” mechanism. Duringtransposition, the PB transposase recognizes transposon-specificinverted terminal repeat sequences (ITRs) located on both ends of thetransposon vector and efficiently moves the contents from the originalsites and efficiently integrates them into TTAA chromosomal sites. Thepowerful activity of the PiggyBac transposon system enables genes ofinterest between the two ITRs in the PB vector to be easily mobilizedinto target genomes.

In some alternatives, a PB contains a drug inducible promoter linked toa polynucleotide coding for a chimeric antigen receptor. In somealternatives, the inducible promoter can be linked to chemokinereceptor, a marker gene, and/or a cytokine. One or more PB transposonscan be employed. In some alternatives, a PB comprises an induciblepromoter linked to a polynucleotide coding for a first chimeric antigenreceptor, another PB comprises an inducible promoter linked to apolynucleotide coding for a second and different chimeric antigenreceptor, and/or a PB comprises an inducible promoter linked to apolynucleotide coding for a chemokine receptor, a chimeric antigenreceptor, and a marker gene. Each element of the constructs is separatedby a nucleic acid, such as that coding for a self-cleaving T2A sequence.In some alternatives each PB differs from one another in the chimericantigen receptor including but not limited to the spacer length andsequence, the intracellular signaling domain, and/or the markersequence. The PB vector can be used with a constitutive lentivirusvector coding for a transcriptional activator for the inducible promoterand constitutive vector comprising the piggyback transposase linked to aconstitutive promoter.

In some alternatives, a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for chimeric antigen receptor comprising aligand binding domain, wherein the ligand binding domain is specific fora ligand, wherein the ligand is a tumor specific molecule, viralmolecule, or any other molecule expressed on a target cell population,wherein the ligand can elicit recognition, modulation, inhibition,and/or elimination by a lymphocyte; a polynucleotide coding for apolypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain; and asecond nucleic acid comprising a second constitutive or induciblepromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the firstand second nucleic acid are in a single lentivirus vector. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor.

In some alternatives, the first nucleic acid further comprises a markergene. In some alternatives, the second nucleic acid further comprisespolynucleotide coding for a second and different chimeric antigenreceptor. The first and second chimeric antigen receptor can differ fromone another in the ligand binding domain, the target antigen, an epitopeof the target antigen, the spacer domain in length and sequence (shortmedium or long), and in the intracellular signaling domains.

In some alternatives, in a single lentivirus construct the first andsecond nucleic acids can be separated by a genomic insulator nucleicacid such as the sea urchin insulator chromatin domain. In otheralternatives, the inducible promoter of the first nucleic acid and theconstitutive promoter of the second nucleic acid are in oppositeorientation.

One or more of these vectors can be used in conjunction with one anotherto transduce target cells and provide for inducible expression of achimeric antigen receptor.

Host Cells and Compositions: T Lymphocyte Populations.

The compositions described herein provide for genetically modified hostcells with the vectors and/or constructs as described herein. In somealternatives, the host cells are CD4+ and/or CD8+ T lymphocytes.

T lymphocytes can be collected in accordance with known techniques andenriched or depleted by known techniques such as affinity binding toantibodies such as flow cytometry and/or immunomagnetic selection. Afterenrichment and/or depletion steps, in vitro expansion of the desired Tlymphocytes can be carried out in accordance with known techniques orvariations thereof that will be apparent to those skilled in the art. Insome alternatives, the T cells are autologous T cells obtained from thepatient.

For example, the desired T cell population or subpopulation can beexpanded by adding an initial T lymphocyte population to a culturemedium in vitro, and then adding to the culture medium feeder cells,such as non-dividing peripheral blood mononuclear cells (PBMC), (e.g.,such that the resulting population of cells contains at least 5, 10, 20,or 40 or more PBMC feeder cells for each T lymphocyte in the initialpopulation to be expanded); and incubating the culture (e.g. for a timesufficient to expand the numbers of T cells). The non-dividing feedercells can comprise gamma-irradiated PBMC feeder cells. In somealternatives, the PBMC are irradiated with gamma rays in the range of3000 to 3600 rads to prevent cell division. In some alternatives, thePBMC are irradiated with gamma rays of 3000, 3100, 3200, 3300, 3400,3500 or 3600 rads or any value of rads between any two endpoints of anyof the listed values to prevent cell division. The order of addition ofthe T cells and feeder cells to the culture media can be reversed ifdesired. The culture can typically be incubated under conditions oftemperature and the like that are suitable for the growth of Tlymphocytes. For the growth of human T lymphocytes, for example, thetemperature will generally be at least 25 degrees Celsius, preferably atleast 30 degrees, more preferably 37 degrees. In some alternatives, thetemperature for the growth of human T lymphocytes is 22, 24, 26, 28, 30,32, 34, 36, 37 degrees Celsius or any other temperature between any twoendpoints of any of the listed values.

The T lymphocytes expanded include CD8⁺ cytotoxic T lymphocytes (CTL)and CD4⁺ helper T lymphocytes that can be specific for an antigenpresent on a human tumor or a pathogen. In some alternatives, the cellsinclude precursor T cells. In some alternatives, the cells arehematopoietic stem cells.

In some alternatives, the expansion method can further comprise addingnon-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells.LCL can be irradiated with gamma rays in the range of 6000 to 10,000rads. In some alternatives, the LCL are irradiated with gamma rays in of6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500 or 10,000 rads or anyamount of rads between two endpoints of any of the listed values. TheLCL feeder cells can be provided in any suitable amount, such as a ratioof LCL feeder cells to initial T lymphocytes of at least 10:1.

In some alternatives, the expansion method can further comprise addinganti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at aconcentration of at least 0.5 ng/ml). In some alternatives, theexpansion method can further comprise adding IL-2 and/or IL-15 to theculture medium (e.g., wherein the concentration of IL-2 is at least 10units/ml).

After isolation of T lymphocytes both cytotoxic and helper T lymphocytescan be sorted into naïve, memory, and effector T cell subpopulationseither before or after expansion.

CD8+ cells can be obtained by using standard methods. In somealternatives, CD8+ cells are further sorted into naïve, central memory,and effector memory cells by identifying cell surface antigens that areassociated with each of those types of CD8+ cells. In some alternatives,memory T cells are present in both CD62L+ and CD62L− subsets of CD8+peripheral blood lymphocytes. PBMC are sorted into CD62L−CD8+ andCD62L+CD8+ fractions after staining with anti-CD8 and anti-CD62Lantibodies. In some alternatives, the expression of phenotypic markersof central memory T_(CM) include CD45RO, CD62L, CCR7, CD28, CD3, and/orCD127 and are negative or low for granzyme B. In some alternatives,central memory T cells are CD45RO+, CD62L+, and/or CD8+ T cells. In somealternatives, effector T_(E) are negative for CD62L, CCR7, CD28, and/orCD127, and positive for granzyme B and/or perforin. In somealternatives, naïve CD8+ T lymphocytes are characterized by theexpression of phenotypic markers of naïve T cells including CD62L, CCR7,CD28, CD3, CD127, and/or CD45RA.

CD4+ T helper cells are sorted into naïve, central memory, and effectorcells by identifying cell populations that have cell surface antigens.CD4+ lymphocytes can be obtained by standard methods. In somealternatives, naïve CD4+ T lymphocytes are CD45RO−, CD45RA+, CD62L+,and/or CD4+ T cells. In some alternatives, central memory CD4+ cells areCD62L+ and/or CD45RO+. In some alternatives, effector CD4+ cells areCD62L− and/or CD45RO−.

Whether a cell or cell population is positive for a particular cellsurface marker can be determined by flow cytometry using staining with aspecific antibody for the surface marker and an isotype matched controlantibody. A cell population negative for a marker refers to the absenceof significant staining of the cell population with the specificantibody above the isotype control, positive refers to uniform stainingof the cell population above the isotype control. In some alternatives,a decrease in expression of one or markers refers to loss of 1 log 10 inthe mean fluorescence intensity and/or decrease of percentage of cellsthat exhibit the marker of at least 20% of the cells, 25% of-the cells,30% of the cells, 35% of the cells, 40% of the cells, 45% of the cells,50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells,70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells,90% of the cell, 95% of the cells, and 100% of the cells or any %between 20 and 100% when compared to a reference cell population. Insome alternatives, a cell population positive for one or markers refersto a percentage of cells that exhibit the marker of at least 50% of thecells, 55% of the cells, 60% of the cells, 65% of the cells, 70% of thecells, 75% of the cells, 80% of the cells, 85% of the cells, 90% of thecell, 95% of the cells, or 100% of the cells or any % between 50 and100% when compared to a reference cell population.

Whether a cell or cell population is positive for a particular cellsurface marker can be determined by flow cytometry using staining with aspecific antibody for the surface marker and an isotype matched controlantibody. A cell population negative for a marker refers to the absenceof significant staining of the cell population with the specificantibody above the isotype control, positive refers to uniform stainingof the cell population above the isotype control. In some alternatives,a decrease in expression of one or markers refers to loss of 1 log 10 inthe mean fluorescence intensity and/or decrease of percentage of cellsthat exhibit the marker of at least 20% of the cells, 25% of-the cells,30% of the cells, 35% of the cells, 40% of the cells, 45% of the cells,50% of the cells, 55% of the cells, 60% of the cells, 65% of the cells,70% of the cells, 75% of the cells, 80% of the cells, 85% of the cells,90% of the cell, 95% of the cells, and 100% of the cells or any %between 20 and 100% when compared to a reference cell population. Insome alternatives, an increase refers to an increase in meanfluorescence intensity and/or to an increase in the number of cells in acell population that are positive for one or a given marker, such as apopulation in which s refers to a percentage of cells that exhibit themarker of at least 50% of the cells, 55% of the cells, 60% of the cells,65% of the cells, 70% of the cells, 75% of the cells, 80% of the cells,85% of the cells, 90% of the cell, 95% of the cells, or 100% of thecells or any % between 50 and 100% exhibit the marker, e.g., whencompared to a reference cell population.

In some alternatives, populations of CD4+ and CD8+ that are antigenspecific can be obtained by stimulating naïve or antigen specific Tlymphocytes with antigen. For example, antigen-specific T cell lines orclones can be generated to Cytomegalovirus antigens by isolating T cellsfrom infected subjects and stimulating the cells in vitro with the sameantigen. Naïve T cells can also be used. Any number of antigens fromtumor cells can be utilized as targets to elicit T cell responses. Insome alternatives, the adoptive cellular immunotherapy compositions areuseful in the treatment of a disease or disorder including a solidtumor, hematologic malignancy, breast cancer or melanoma.

Modification of T Lymphocyte Populations.

In some alternatives it can be desired to introduce functional genesinto the T cells to be used in immunotherapy in accordance with thepresent disclosure. For example, the introduced gene or genes canimprove the efficacy of therapy by promoting the viability and/orfunction of transferred T cells; or they can provide a genetic marker topermit selection and/or evaluation of in vivo survival or migration; orthey can incorporate functions that improve the safety of immunotherapy,for example, by making the cell susceptible to controlled expression ofthe transgene. This can be carried out in accordance with knowntechniques that will be apparent to those skilled in the art based uponthe present disclosure.

In some alternatives, T cells are modified with a vector coding for druginducible chimeric receptors as described herein. In some alternatives,cells are modified with a vector comprising a polynucleotide coding fora chimeric antigen receptor under control of an inducible promoter. Inother alternatives, cells are modified with a vector comprising apolynucleotide coding for a cytokine, chemokine receptor, a gene thatregulates apoptosis, or a gene that modulates checkpoint signaling underthe control of an inducible promoter. In some alternatives, the T cellsare obtained from the subject to be treated, in other alternatives, thelymphocytes are obtained from allogeneic human donors, preferablyhealthy human donors. In some alternatives, the modulation of checkpointsignaling inhibits negative checkpoint regulators. In some alternatives,the negative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3.

Chimeric receptors can be constructed with a specificity for any cellsurface marker by utilizing antigen binding fragments or antibodyvariable domains of, for example, antibody molecules. The antigenbinding molecules can be linked to one or more cell signaling modules.In some alternatives, cell signaling modules include CD3 transmembranedomains, CD3 intracellular signaling domains, and/or CD28 transmembranedomains. In some alternatives, the intracellular signaling domaincomprises a CD28 transmembrane and signaling domain linked to a CD3 zetaintracellular domain. In some alternatives, a chimeric receptor can alsoinclude a transduction marker such as tEGFR.

In some alternatives, the same or a different chimeric receptor can beintroduced into each of population of CD4+ and/or CD8+ T lymphocytes. Insome alternatives, the chimeric receptor in each of these populationshas a ligand binding domain that specifically binds to the same ligandon the tumor or infected cell or a different antigen or epitope. Thecellular signaling modules can differ. In some alternatives, theintracellular signaling domain of the CD8+ cytotoxic T cells is the sameas the intracellular signaling domain of the CD4+ helper T cells. Inother alternatives, the intracellular signaling domain of the CD8+cytotoxic T cells is different than the intracellular signaling domainof the CD4+ helper T cells.

In some alternatives each of the CD4 or CD8 T lymphocytes can be sortedinto naïve, central memory, effector memory or effector cells prior totransduction, as described herein. In some alternatives, each of the CD4or CD8 T lymphocytes can be sorted into naïve, central memory, effectormemory, or effector cells after transduction.

As described herein, in some alternatives, naïve CD4+ cells are CD45RO−,CD45RA+, CD62L+, and/or CD4+ positive T cells. In some alternatives,central memory CD4+ cells are CD62L positive and/or CD45RO positive. Insome alternatives, effector CD4+ cells are CD62L negative and/or CD45ROpositive. Each of these populations can be independently modified with achimeric receptor.

As described, in some alternatives, memory T cells are present in bothCD62L+ and CD62L− subsets of CD8+ peripheral blood lymphocytes. PBMC aresorted into CD62L−CD8+ and CD62L+CD8+ fractions after staining withanti-CD8 and anti-CD62L antibodies. In some alternatives, the expressionof phenotypic markers of central memory T cells (TCM) include CD62L,CCR7, CD28, CD3, and/or CD127 and are negative or low for granzyme B. Insome alternatives, central memory T cells are CD45RO+, CD62L+, and/orCD8+ T cells. In some alternatives, effector T cells (T_(E)) arenegative for CD62L, CCR7, CD28, and/or CD127, and positive for granzymeB and/or perforin. In some alternatives, naïve CD8+ T lymphocytes arecharacterized by CD8+, CD62L+, CD45RO+, CCR7+, CD28+ CD127+, and/orCD45RO+. Each of these populations can be independently modified with achimeric receptor.

Various transduction techniques have been developed which utilizerecombinant infectious virus particles for gene delivery. Thisrepresents a currently preferred approach to the transduction of Tlymphocytes of the present invention. The viral vectors, which have beenused in this way include virus vectors derived from simian virus 40,adenoviruses, adeno-associated virus (AAV), lentiviral vectors, and/orretroviruses. Thus, gene transfer and expression methods are numerousbut essentially function to introduce and express genetic material inmammalian cells. Several of the above techniques have been used totransduce hematopoietic or lymphoid cells, including calcium phosphatetransfection, protoplast fusion, electroporation, and infection withrecombinant adenovirus, adeno-associated virus and retrovirus vectors.Primary T lymphocytes have been successfully transduced byelectroporation and by retroviral or lentiviral infection.

Retroviral and lentiviral vectors provide a highly efficient method forgene transfer into eukaryotic cells. Moreover, retroviral or lentiviralintegration takes place in a controlled fashion and results in thestable integration of one or a few copies of the new genetic informationper cell.

It is contemplated that overexpression of a stimulatory factor (forexample, a lymphokine or a cytokine) can be toxic to the treatedindividual. Therefore, it is within the scope of the invention toinclude gene segments that cause the T cells of the invention to besusceptible to negative selection in vivo. By “negative selection” ismeant that the infused cell can be eliminated as a result of a change inthe in vivo condition of the individual. The negative selectablephenotype can result from the insertion of a gene that conferssensitivity to an administered agent, for example, a compound. Negativeselectable genes are known in the art, and include, inter alia thefollowing: the Herpes simplex virus type I thymidine kinase (HSV-I TK)gene, which confers ganciclovir sensitivity; the cellular hypoxanthinephosphribosyltransferase (HPRT) gene, the cellular adeninephosphoribosyltransferase (APRT) gene, and bacterial cytosine deaminase.

In some alternatives it can be useful to include in the T cells apositive marker that enables the selection of cells of the negativeselectable phenotype in vitro. The positive selectable marker can be agene that upon being introduced into the host cell expresses a dominantphenotype permitting positive selection of cells carrying the gene.Genes of this type are known in the art, and include, inter alia,hygromycin-B phosphotransferase gene (hph), which confers resistance tohygromycin B, the amino glycoside phosphotransferase gene (neo or aph)from Tn5, which codes for resistance to the antibiotic G418, thedihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA),and/or the multi-drug resistance (MDR) gene.

A variety of methods can be employed for transducing T lymphocytes, asis well known in the art. In some alternatives, transduction is carriedout using lentiviral vectors.

In some alternatives, CD4+ and CD8+ cells each can separately bemodified with an expression vector encoding a chimeric receptor to formdefined populations. In some alternatives, cells can be separatelymodified with a vector comprising a polynucleotide under the control ofa constitutive promoter and a vector comprising a polynucleotide codingfor a cytokine or chemokine receptor under control of an induciblepromoter.

In some alternatives, these cells are then further sorted intosubpopulations of naïve, central memory and effector cells as describedabove, by sorting for cell surface antigens unique to each of those cellpopulations. In addition, CD4+ or CD8+ cell populations can be selectedby their cytokine profile or proliferative activities. For example, CD4+T lymphocytes that have enhanced production of cytokines such as IL-2,IL-4, IL-10, TNFα, and/or IFNγ, as compared to sham transduced cells ortransduced CD8+ cells when stimulated with antigen can be selected. Inother alternatives, naïve or central memory CD4+ T cells that haveenhanced production of IL-2 and/or TNFα are selected. Likewise, CD8+cells that have enhanced IFNγ production are selected, as compared tosham transduced CD8+ cells.

In some alternatives, CD4+ and CD8+ cells are selected that arecytotoxic for antigen bearing cells. In some alternatives, CD4+ areexpected to be weakly cytotoxic as compared to CD8+ cells. In apreferred alternative, transduced lymphocytes, such as CD8+ centralmemory cells, are selected that provide for tumor cell killing in vivousing an animal model established for the particular type of cancer.

In yet other alternatives, transduced chimeric receptor expressing Tcells are selected that can persist in vivo using an animal modelestablished for the particular type of cancer. In some alternatives,transduced chimeric receptor CD8+ central memory cells with a shortspacer region have been shown to persist in vivo after introduction intothe animal for 3 days or more, 10 days or more, 20 days or more, 30 daysor more, 40 days or more, or 50 days or more.

The disclosure contemplates that combinations of CD4+ and CD8+ T cellswill be utilized in the compositions. In one alternative, combinationsof chimeric receptor transduced CD4+ cells can be combined with chimericreceptor transduced CD8+ cells of the same ligand specificity orcombined with CD8+ T cells that are specific for a distinct tumorligand. In other alternatives, chimeric receptor transduced CD8+ cellsare combined with chimeric receptor transduced CD4+ cells specific for adifferent ligand expressed on the tumor. In yet another alternative,chimeric receptor modified CD4+ and CD8+ cells are combined. In somealternatives CD8+ and CD4+ cells can be combined in different ratios forexample, a 1:1 ratio of CD8+ and CD4+, a ratio of 10:1 of CD8+ to CD4+,or a ratio of 100:1 of CD8+ to CD4+, or any other ratio of CD8+ to CD4+that is between any of the listed ratios. In some alternatives, thecombined population is tested for cell proliferation in vitro and/or invivo, and the ratio of cells that provides for proliferation of cells isselected.

After transduction and/or selection for chimeric receptor bearing cells,the cell populations are preferably expanded in vitro until a sufficientnumber of cells are obtained to provide for at least one infusion into ahuman subject, typically around 10⁴ cells/kg to 10⁹ cells/kg In somealternatives, the transduced cells are cultured in the presence ofantigen bearing cells, anti CD3, anti CD28, and IL 2, IL-7, IL 15, orIL-21 or combinations thereof.

In some alternatives, CD4+ and CD8+ cells that proliferate in responseto cytokine stimulation, antigen or tumor targets in vitro or in vivoare selected. For example, CD4+ or CD8+ transduced cells thatproliferate vigorously when stimulated with antiCD3 and/or anti-CD28 areselected. In some alternatives, stimulation of transduced cells providesfor enhanced transgene expression in the presence of an inducer (e.g.drug) of those trans genes under the control of an inducible promoter.

Each of the subpopulations of CD4+ and CD8+ cells can be combined withone another. In a specific alternative, modified naïve or central memoryCD4+ cells are combined with modified central memory CD8+ T cells toprovide a synergistic cytotoxic effect on antigen bearing cells, such astumor cells.

Compositions.

The disclosure provides for an adoptive cellular immunotherapycomposition comprising a genetically modified T lymphocyte cellpreparation as described herein. In some alternatives, the T lymphocytecell preparation comprises CD4+ T cells that have a chimeric receptorcomprising an extracellular antibody variable domain specific for aligand associated with the disease or disorder, a spacer region, atransmembrane domain, and an intracellular signaling domain of a T cellreceptor or other receptors under the control of a drug induciblepromoter as described herein. In other alternatives, an adoptivecellular immunotherapy composition further comprises a chimeric receptormodified tumor-specific CD8+ cytotoxic T lymphocyte cell preparationthat provides a cellular immune response, wherein the cytotoxic Tlymphocyte cell preparation comprises CD8+ T cells that have a chimericreceptor comprising an extracellular single chain antibody specific fora ligand associated with the disease or disorder, a spacer region, atransmembrane domain, and an intracellular signaling domain of a T cellreceptor under the control of a drug inducible promoter as describedherein. In some alternatives, the chimeric receptor modified T cellpopulation of the disclosure can persist in vivo for at least 3 days orlonger. In an alternative, each of these populations can be combinedwith one another or other cell types to provide a composition.

In some alternatives, the CD4+ T helper lymphocyte cell is naïve CD4+ Tcells, central memory CD4+ T cells, effector memory CD4+ T cells, orbulk CD4+ T cells. In some alternatives, CD4+ helper lymphocyte cell isa naïve CD4+ T cell, wherein the naïve CD4+ T cell comprises a CD45RO−,CD45RA+, and/or is a CD62L+ CD4+ T cell.

In some alternatives, the CD8+ T cytotoxic lymphocyte cell is a naïveCD8+ T cell, central memory CD8+ T cell, effector memory CD8+ T celland/or bulk CD8+ T cell. In some alternatives, the CD8+ cytotoxic Tlymphocyte cell is a central memory T cell, wherein the central memory Tcell comprises a CD45RO+, CD62L+, and/or CD8+ T cell. In yet otheralternatives, the CD8+ cytotoxic T lymphocyte cell is a central memory Tcell and the CD4+ helper T lymphocyte cell is a naïve or central memoryCD4+ T cell.

In some alternatives, the compositions comprise T cell precursors. Insome alternatives, the compositions comprise hematopoietic stem cells.In some alternatives, the composition comprises a host cell wherein thehost cell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells or a CD4+ T helper lymphocytecell that is selected from the group consisting of naïve CD4+ T cells,central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+T cells and a second host cell, wherein the second host cell is aprecursor T cell. In some alternatives, the precursor T cell is ahematopoietic stem cell.

Methods.

The disclosure provides methods of making adoptive immunotherapycompositions and uses or methods of using these compositions forperforming cellular immunotherapy in a subject having a disease ordisorder. In some alternatives, a method of manufacturing thecompositions comprises obtaining a modified naïve or central memory CD4+T helper cell, wherein the modified helper T lymphocyte cell preparationcomprises CD4+ T cells that have a chimeric receptor comprising a ligandbinding domain specific for a tumor cell surface molecule, a spacerdomain, a transmembrane domain, and an intracellular signaling domainunder control of an inducible promoter as described herein. In otheralternatives, CD4+ cells have a cytokine or chemokine receptor under thecontrol of an inducible promoter.

In another alternative, a method further comprises obtaining a modifiedCD8+ central memory T cell, wherein the modified central memory CD8 Tlymphocyte cell preparation comprises CD8+ cells that have a chimericreceptor comprising a ligand binding domain, wherein the ligand bindingdomain is specific for a ligand, wherein the ligand is a tumor specificmolecule, viral molecule, or any other molecule expressed on a targetcell population, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte, a spacer domain, atransmembrane domain, and an intracellular signaling domain undercontrol of the inducible promoter as described herein. In otheralternatives, CD4+ cells have a cytokine or chemokine receptor under thecontrol of an inducible promoter.

The drug inducible promoter in both modified CD4+ T cells and modifiedCD8+ cytotoxic T cells can be the same or different. In somealternatives, in one population of cells the promoter linked to thechimeric antigen receptor is a constitutive promoter and in the otherpopulation it is an inducible promoter. For example, modified CD4+ Tcells that have a chimeric receptor comprising a ligand binding domainspecific for a tumor cell surface molecule, a spacer domain, atransmembrane domain, and an intracellular signaling domain undercontrol of an constitutive promoter, while the CD8+ cytotoxic T cellcomprises CD8+ cells that have a chimeric receptor comprising a ligandbinding domain specific for a tumor cell surface molecule, a spacerdomain, a transmembrane domain, and an intracellular signaling domainunder control of the inducible promoter.

In some alternatives, the polynucleotide can code for a chimeric antigenreceptor that differs in the CD4+ versus the CD8+ cell population. Thedifference between the two constructs can include the specificity oraffinity of the ligand binding domain for an antigen or epitope, thelength and sequence of the spacer region, and the intracellularsignaling components.

The preparation of the CD4+ and CD8+ cells that are modified with achimeric receptor is described throughout this disclosure. Antigenspecific T lymphocytes can be obtained from a patient having the diseaseor disorder or can be prepared by in vitro stimulation of T lymphocytesin the presence of antigen. Subpopulations of CD4+ and/or CD8+ Tlymphocytes that are not selected for antigen specificity can also beisolated as described herein and combined in the methods ofmanufacturing.

In some alternatives, the combination of cell populations can beevaluated for uniformity of cell surface makers, the ability toproliferate through at least two generations, to have a uniform celldifferentiation status. Quality control can be performed by co-culturinga cell line expressing the target ligand with chimeric receptor modifiedT cells and the drug that induces expression of the chimeric antigenreceptor to determine if the chimeric receptor modified T cellsrecognize the cell line using cytotoxicity, proliferation, or cytokineproduction assays in the presence of the inducer that are known in thefield. Cell differentiation status and cell surface markers on thechimeric receptor modified T cells can be determined by flow cytometry.In some alternatives, the markers and cell differentiation status on theCD8+ cells include CD3, CD8, CD62L, CD28, CD27, CD69, CD25, PD-1,CTLA-4, CD45RO, and/or CD45RA. In some alternatives, the markers and thecell differentiation status on the CD4+ cells include CD3, CD4, CD62L,CD28, CD27, CD69, CD25, PD-1, CTLA-4 CD45RO, and/or CD45RA.

In some alternatives, the chimeric receptor modified T cells asdescribed herein are able to persist in vivo for at least 3 days, or atleast 10 days. In some alternatives, the chimeric receptor modified Tcells as described herein, can proliferate in vivo through at least 2,or at least 3 generations as determined by CFSE dye dilution.Proliferation and persistence of the chimeric receptor modified T cellscan be determined by using an animal model of the disease or disorderand administering the cells and determining persistence and/orproliferative capacity of the transferred cells. In other alternatives,proliferation and activation can be tested in vitro by going throughmultiple cycles of activation with antigen bearing cells.

The disclosure also provides methods of performing cellularimmunotherapy in a subject having a disease or disorder comprising:administering a composition of lymphocytes expressing a chimericreceptor under the control of a drug inducible promoter as describedherein, and administering the drug.

In some alternatives, the drug is tamoxifen, variants, derivatives,pharmaceutical salts, solvates, and hydrates thereof as describedherein. In some alternatives, the drug is delivered prior to, at thesame time as the composition, or at later time points after thecomposition has been administered.

In some alternatives, the drug is administered with the composition, andif a toxic effect of the composition is observed the drug is withdrawnuntil the toxic effects diminish. After the symptoms of toxicitydiminish, the drug is administered again. In some alternatives, a drugcan be administered again once symptoms of toxicity diminish.

In some alternatives, the drug is administered with the composition butonce the subject has a decrease in the tumor load or cancer cells, thedrug is withdrawn for a period of time to allow the modified cells torest and if there is no activity of the modified cells, the modifiedcells are not needed because of remission of the cancer.

In other alternatives, a method comprises administering to the subject agenetically modified cytotoxic T lymphocyte cell preparation thatprovides a cellular immune response, wherein the cytotoxic T lymphocytecell preparation comprises CD8+ T cells that have a chimeric receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte, a spacer domain, atransmembrane domain, and an intracellular signaling domain under thecontrol of a drug inducible promoter as described herein, and/or agenetically modified helper T lymphocyte cell preparation that elicitsdirect tumor recognition and augments the genetically modified cytotoxicT lymphocyte cell preparations ability to mediate a cellular immuneresponse, wherein the helper T lymphocyte cell preparation comprisesCD4+ T cells that have a chimeric receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte, a spacer domain, a transmembrane domain, and anintracellular signaling domain under control of a constitutive or druginducible promoter as described herein and administering the drug thatinduces the inducible promoter. In some alternatives, the administeringof the drug is performed after administering of the composition or hostcells, wherein administering is performed 1 day, 2 days, 3 days, 4 days,5 days, 6 days, 7 days, 2 weeks, 4 weeks or two months, or any time inbetween any two values of time listed.

In other alternatives, a method comprises administering to the subject agenetically modified cytotoxic T lymphocyte cell preparation thatprovides a cellular immune response, wherein the cytotoxic T lymphocytecell preparation comprises CD8+ T cells that have a chimeric receptorcomprising a ligand binding domain specific for a tumor cell surfacemolecule, a spacer domain, a transmembrane domain, and an intracellularsignaling domain under the control of a constitutive promoter asdescribed herein, and/or a genetically modified helper T lymphocyte cellpreparation that elicits direct tumor recognition and augments thegenetically modified cytotoxic T lymphocyte cell preparations ability tomediate a cellular immune response, wherein the helper T lymphocyte cellpreparation comprises CD4+ T cells that have a chimeric receptorcomprising a ligand binding domain specific for a tumor cell surfacemolecule, a spacer domain, a transmembrane domain, and an intracellularsignaling domain under control of a constitutive or drug induciblepromoter as described herein and administering the drug that induces theinducible promoter. In some alternatives, the tumor specific molecule isa tumor surface molecule.

In other alternatives, a method comprises administering to the subject agenetically modified cytotoxic T lymphocyte cell preparation thatprovides a cellular immune response, wherein the cytotoxic T lymphocytecell preparation comprises CD8+ T cells that express a cytokine,chemokine receptor, a polypeptide that regulates apoptosis, and/or apolypeptide that modulates checkpoint signaling under the control of aninducible promoter as described herein, and/or a genetically modifiedhelper T lymphocyte cell preparation that elicits direct tumorrecognition and augments the genetically modified cytotoxic T lymphocytecell preparations ability to mediate a cellular immune response, whereinthe helper T lymphocyte cell preparation comprises CD4+ T cells thatexpress a cytokine, chemokine receptor, a polypeptide that regulatesapoptosis, and/or a polypeptide that modulates checkpoint signalingunder control of a constitutive or drug inducible promoter as describedherein and administering the drug that induces the inducible promoter.In some alternatives, one or more of the cell populations expresses achimeric antigen receptor under the control of a constitutive promoter.In some alternatives, the modulation of checkpoint signaling inhibitsnegative checkpoint regulators. In some alternatives, the negativecheckpoint regulator comprises VISTA, LAG-3 and/or TIM3.

An effective amount of the drug for induction is an amount of the drugthat provides for induction of the chimeric antigen receptor in at least5%, at least 10%, at least 20%, at least 30%, at least 40%, at least50%, at least 60%, at least 70%, at least 80%, at least 90%, at least100% or any number in between any of the listed percent values of thetransduced cells.

Another alternative describes a method of performing cellularimmunotherapy in a subject having a disease or disorder comprising:analyzing a biological sample of the subject for the presence of atarget molecule associated with the disease or disorder andadministering the adoptive immunotherapy compositions described hereinand administering the drug that induces the inducible promoter, whereinthe chimeric receptor specifically binds to the target molecule.

Subjects that can be treated by the present invention are, in general,human and other primate subjects, such as monkeys and apes forveterinary medicine purposes. The subjects can be male or female and canbe any suitable age, including infant, juvenile, adolescent, adult, andgeriatric subjects.

The methods are useful in the treatment or inhibition of, for example,hematologic malignancy, melanoma, breast cancer, brain cancer, and otherepithelial malignancies or solid tumors. In some alternatives, themolecule associated with the disease or disorder is an orphan tyrosinekinase receptor ROR1, Her2, EGFR, CE7, hB7H3, CD19, CD20, CD22,mesothelin, CEA, or a hepatitis B surface antigen.

Subjects that can be addressed using the methods described hereininclude subjects identified or selected as having cancer, including butnot limited to colon, lung, liver, breast, renal, prostate, ovarian,skin (including melanoma), bone, and brain cancer, etc. Suchidentification and/or selection can be made by clinical or diagnosticevaluation. In some alternatives the tumor associated antigens ormolecules are known, such as melanoma, breast cancer, brain cancer,squamous cell carcinoma, colon cancer, leukemia, myeloma, and/orprostate cancer. In other alternatives the tumor associated moleculescan be targeted with genetically modified T cells expressing anengineered chimeric receptor. Examples include but are not limited to Bcell lymphoma, breast cancer, brain cancer, prostate cancer, and/orleukemia.

Cells prepared as described above can be utilized in methods andcompositions for adoptive immunotherapy in accordance with knowntechniques, or variations thereof that will be apparent to those skilledin the art based on the instant disclosure.

In some alternatives, the cells are formulated by first harvesting themfrom their culture medium, and then washing and concentrating the cellsin a medium and container system suitable for administration (a“pharmaceutically acceptable” carrier) in a treatment-effective amount.Suitable infusion medium can be any isotonic medium formulation,typically normal saline, Normosol R (Abbott) or Plasma-Lyte A (Baxter),but also 5% dextrose in water or Ringer's lactate can be utilized. Theinfusion medium can be supplemented with human serum albumin, fetalbovine serum or other human serum components.

In some alternatives, a treatment or inhibitory effective amount ofcells in the composition is a transduced CD4 or CD8 cell or at least 2cell subsets (for example, 1 CD8+ central memory T cell subset and 1CD4+ helper T cell subset) or is more typically greater than 10² cells,and up to 10⁶, up to and including 10⁸ or 10⁹ cells and can be more than10¹⁰ cells. The number of cells will depend upon the ultimate use forwhich the composition is intended as will the type of cells includedtherein. For example, if cells that are specific for a particularantigen are desired, then the population will contain greater than 70%,generally greater than 80%, 85% and 90-95% of such cells. For usesprovided herein, the cells are generally in a volume of a liter or less,can be 500 mls or less, even 250 mls or 100 mls or less or a volume inbetween any two listed volume values. Hence the density of the desiredcells is typically greater than 10⁴ cells/ml and generally is greaterthan 10⁷ cells/ml, generally 10⁸ cells/ml or greater. The clinicallyrelevant number of immune cells can be apportioned into multipleinfusions that cumulatively equal or exceed 10⁶, 10⁷, 10⁸, 10⁸, 10⁹,10¹⁰ or 10¹¹ cells or any amount of cells defined between any twoendpoints of any of the listed values.

In some alternatives, the lymphocytes of the invention can be used toconfer immunity to individuals. By “immunity” is meant a lessening ofone or more physical symptoms associated with a response to infection bya pathogen, or to a tumor, to which the lymphocyte response is directed.The amount of cells administered is usually in the range present innormal individuals with immunity to the pathogen. Thus, the cells areusually administered by infusion, with each infusion in a range of from2 cells, up to at least 10⁶ to 3×10¹⁰ cells, preferably in the range ofat least 10⁷ to 10⁹ cells. The T cells can be administered by a singleinfusion, or by multiple infusions over a range of time. However, sincedifferent individuals are expected to vary in responsiveness, the typeand amount of cells infused, as well as the number of infusions and thetime range over which multiple infusions are given are determined by theattending physician, and can be determined by routine examination. Thegeneration of sufficient levels of T lymphocytes (including cytotoxic Tlymphocytes and/or helper T lymphocytes) is readily achievable using therapid expansion method of the present invention, as exemplified herein.

In some alternatives, a composition as described herein is administeredto an identified or selected subject, such as a subject identified orselected as having melanoma, breast cancer, brain cancer, squamous cellcarcinoma, colon cancer, leukemia, myeloma, and/or prostate cancer,intravenously, intraperitoneally, intratumorly, into the bone marrow,into the lymph node, and/or into cerebrospinal fluid. In somealternatives, the chimeric receptor engineered compositions aredelivered to the site of the tumor. Alternatively, the compositions asdescribed herein can be combined with a compound that targets the cellsto the tumor or the immune system compartments and avoid sites such asthe lung.

In some alternatives, the compositions as described herein areadministered with chemotherapeutic agents and/or immunosuppressants. Inan alternative, a patient is first administered a chemotherapeutic agentthat inhibits or destroys other immune cells followed by thecompositions described herein. In some cases, chemotherapy can beavoided entirely.

In some alternatives, a method comprising administering the modified Tcells as described herein in combination with the inducer (e.g.inducible drug) until the tumor burden is diminished. Once the tumorburden is diminished, the inducer drug can be withdrawn in order toswitch the expression of the chimeric antigen receptor off and decreasethe number of T cell expressing the receptor. In other alternatives, theinducer drug can be administered at different time in order to switchthe expression of the chimeric antigen receptor on in the event of arelapse or increase in tumor growth.

In other alternatives, the inducer drug can be given for a period oftime of days, weeks, or months, and then withdrawn for days, weeks ormonths, followed by re-administration of the inducer drug for days,weeks or months to allow for cycling of the expression of the chimericantigen receptor to avoid anergy or nonresponsiveness due to chronicstimulation of the cells.

Vector Construction and Preparation of Dual Packaged Lentivirus.

An inducible lentiviral vector encoding7xHBD/mE1b-CD19t-her2t-T2A-epHIV7) was constructed. CD19t specificchimeric receptors were constructed using: (1) the VL and VH chainsegments of the CD19-specific mAb FMC63 (SEQ ID NO: 3), linked by a(G₄S)₃ linker (SEQ ID NO: 12) peptide (VL-linker-VH); (2) a spacerdomain derived from IgG4-Fc Hinge only (12 AA encoded by (SEQ ID NO:4)). Spacers contained a S→P substitution within the Hinge domainlocated at position 108 of the native IgG4-Fc protein; the 27 AAtransmembrane domain of human CD28 (Uniprot Database: P10747, (SEQ IDNO: 14)); (4) a signaling module comprising either (i) the 41 AAcytoplasmic domain of human CD28 with an LL→GG substitution located atposition 186-187 of the native CD28 protein (SEQ ID NO: 14); and/or (ii)the 42 AA cytoplasmic domain of human 4-1BB (Uniprot Database: Q07011,(SEQ ID NO: 15)); linked to (iii) the 112 AA cytoplasmic domain ofisoform 3 of human CD3ζ (Uniprot Database: P20963, (SEQ ID NO: 16)).

The nucleic acid sequences coding for the CD19t were linked withsequences coding for Her2t (SEQ Id NO: 44); and the self-cleaving T2Asequence (SEQ ID NO: 8).

A conditional lentiviral vector encoding 7xHBD/mEF1αp-ZsGreen-epHIV7 wasconstructed. The synthetic promoter 7xHBD/mEF1αp was constructed bycombining seven minimal hepatocyte nuclear family-1 (HNF-1) bindingsites cloned from the human albumin promoter and the huEF1α promoterTATA box and has a sequence of (SEQ ID NO: 41). In this way, only in thepresence of tamoxifen does binding of HEA-3 to 7xHBD/EF1mp promoterinduce the “ON” state of transgene expression.

A conditional lentiviral vector encoding7xHBD/mEF1αp-CD19t-T2A-DHI-Rdm_epHIV7 was constructed. CD19t specificchimeric receptors were constructed using: (1) the VL and VH chainsegments of the CD19-specific mAb FMC63 (SEQ ID NO: 3), linked by a(G₄S)₃ linker (SEQ ID NO: 12) peptide (VL-linker-VH); (2) a spacerdomain derived from IgG4-Fc Hinge only (12 AA encoded by (SEQ ID NO:4)). Spacers contained a S→P substitution within the Hinge domainlocated at position 108 of the native IgG4-Fc protein; the 27 AAtransmembrane domain of human CD28 (Uniprot Database: P10747, (SEQ IDNO: 14)); (4) a signaling module comprising either (i) the 41 AAcytoplasmic domain of human CD28 with an LL→GG substitution located atposition 186-187 of the native CD28 protein (SEQ ID NO: 14); and/or (ii)the 42 AA cytoplasmic domain of human 4-1BB (Uniprot Database: Q07011,(SEQ ID NO: 15)); linked to (iii) the 112 AA cytoplasmic domain ofisoform 3 of human CD3ζ (Uniprot Database: P20963, (SEQ ID NO: 16)).

The nucleic acid sequences coding for the CD19t were linked withsequences coding for the self-cleaving T2A sequence (SEQ ID NO: 8); andDHFRdm (SEQ ID NO: 46)

The transcriptional regulator, HEA-3, is a chimeric transcription factorcomposed of human subunits including the N-terminal DNA binding domainof Hepatocyte Nuclear Factor 1-alpha (HNF-1α) fused in frame to themutant tamoxifen-specific ligand binding domain of the estrogen receptorligand binding domain (ER-LBD), that is in turn fused to the p65activation domain of NF-κβ (p65). In the absence of tamoxifen, HEA-3 isexcluded from the nucleus by binding of cytosolic heat-shock protein 90(HSP90) to the tamoxifen binding active site and transgene expression isin the “OFF” state. Nanomolar concentrations of cytosolic tamoxifenactively outcompete HSP90 for ER-LBD binding, resulting in HEA-3translocation to the nucleus. Upon nuclear translocation, HEA-3 isreadily available to bind its restricted synthetic promoter.Transcriptional responsiveness to HEA-3 in the presence of tamoxifen isachieved when transgenes are placed behind an HEA-3 responsive syntheticpromoter (7xHBD/EF1mp).

A constitutive construct was constructed with a constitutive promoterEF-1α linked to a polynucleotide coding for a transcriptional activatorHEA3 (SEQ ID NO: 39) and a marker sequence EGFRt (SEQ ID NO: 9).

Human codon-optimized nucleotide sequences encoding each transgene weresynthesized (LifeTechnologies, Carlsbad, Calif.) and cloned into theepHIV7 lentiviral vector using NheI and Not1 restriction sites. TheepHIV7 lentiviral vector had been derived from the pHIV7 vector byreplacing the cytomegalovirus promoter of pHIV7 with an EF-1 promoter.

The inducible CD19 chimeric receptor-encoding and the constitutivelentivirus was produced in 293T cells co-transfected with the lentiviralvector and the packaging vectors pCHGP-2, pCMV-Rev2 and pCMV-G usingCalphos transfection reagent (Clontech). Medium was changed 16 hoursafter transfection, and lentivirus collected after 24, 48 and 72 hours.

Generation of Jurkat T-Cell Lines Expressing the CD19 Chimeric Receptorsand ZsGreen when Induced with Tamoxifen.

Jurkat cells were transduced with lentiviral supernatant (MOI=3)supplemented with 1 μg/mL polybrene (Millipore) on day 3 afteractivation by centrifugation at 2,100 rpm for 45 minutes at 32° C. Tcells were expanded in RPM1, 10% human serum, 2 mM L-glutamine and 1%penicillin-streptomycin (CTL medium), supplemented with recombinanthuman (rh) 1L-2 to a final concentration of 50 U/mL every 48 hours.

Additional Alternatives.

In some alternatives, system for inducible expression of a chimericantigen receptor is provided, wherein the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora chimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. . In some alternatives, the spacer is optimized for increasedT cell proliferation and/or cytokine production in response to theligand as compared to a reference chimeric receptor. In somealternatives, the drug is tamoxifen and/or its metabolites. In somealternatives, the first promoter comprises a nucleic acid sequence ofSEQ ID NO: 41. In some alternatives, the second promoter is aconstitutive promoter. In some alternatives, the second promoter is theEF1αp. In some alternatives, the transcriptional activator comprises asequence of SEQ ID NO: 40. In some alternatives, the first nucleic acidfurther comprises a first vector and the second nucleic acid furthercomprises a second vector. In some alternatives, both vectors arepackaged in a viral vector. In some alternatives, the viral vector is alentivirus. In some alternatives, the first and second nucleic acidcomprise a vector. In some alternatives, the first nucleic acid furthercomprises a nucleic acid sequence coding for a selectable marker. Insome alternatives, the second nucleic acid further comprises a nucleicacid coding for a selectable marker.

In some alternatives, a system for inducible expression of chimericantigen receptor comprises: a first nucleic acid comprising a firstpromoter inducible by a drug, wherein the first nucleic acid is operablylinked to a polynucleotide coding for a cytokine, a chemokine, apolypeptide that regulates apoptosis and/or a polypeptide that modulatescheckpoint signaling; and a second nucleic acid comprising a secondpromoter operably linked to a nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the secondpromoter is constitutive or inducible. In an exemplary alternative, thesecond nucleic acid further comprises a polynucleotide coding for achimeric antigen receptor, under the control of a constitutive promoter.

In another aspect, the present disclosure provides compositions toconfer and/or augment immune responses mediated by cellularimmunotherapy, such as by adoptively transferring tumor-specific, subsetspecific genetically modified CD4+ T cells, wherein the CD4+ T cellsconfer and/or augment the ability of CD8+ T cells to sustain anti-tumorreactivity and increase and/or maximize tumor-specific proliferation. Insome alternatives, the CD4+ cells are genetically modified to express achimeric receptor nucleic acid and/or chimeric receptor polypeptideunder the control of a regulated promoter as described herein.

In another aspect, the present disclosure provides compositions toconfer and/or augment immune responses mediated by cellularimmunotherapy, such as by adoptively transferring tumor-specific, subsetspecific genetically modified CD8+ T cells. In some alternatives, theCD8+ T cells express a chimeric receptor nucleic acid and/or chimericreceptor polypeptide under the control of a regulated promoter, asdescribed herein.

In one alternative, the present invention provides a method ofperforming cellular immunotherapy in a subject having a disease ordisorder by administering to the subject a genetically modified Tlymphocyte cell preparation that provides a cellular immune response andadministering a drug that induces a transgene in the geneticallymodified T lymphocyte cells.

In some alternatives, the genetically modified CD8+ and geneticallymodified CD4+ cell population are co-administered. In some alternatives,the T cells are autologous or allogeneic T cells. Various modificationsof the above method are possible. For example, the chimeric receptorthat is expressed by the CD4+ T cell and the CD8+ T cell can be the sameor different.

In some alternatives, a system for inducible expression of a chimericantigen receptor is provided, wherein the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora chimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte;a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain; and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the second promoter is constitutive orinducible. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thespacer is optimized for increased T cell proliferation and/or cytokineproduction in response to the ligand as compared to a reference chimericreceptor.

In some alternatives, a system for inducible expression of chimericantigen receptor is provided, wherein the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora cytokine, a chemokine receptor, a polypeptide that regulatesapoptosis, or a polypeptide that modulates checkpoint signaling; and b)a second nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the polypeptide that modulatescheckpoint signaling inhibits negative checkpoint regulators. In somealternatives, the negative checkpoint regulator comprises VISTA, LAG-3and/or TIM3. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor; a polynucleotide codingfor a transmembrane domain. In some alternatives, the first promoter isin opposite orientation to the second promoter. In some alternatives,the ligand binding domain is an antibody fragment. In some alternatives,the ligand binding domain is single chain variable fragment. In somealternatives, the tumor specific molecule is CD19, CD20, CD22, CD23,CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2,GD-2, or MAGE A3 TCR or combinations thereof. In some alternatives, thesecond promoter is an inducible promoter or a constitutive promoter.

In some alternatives, a chimeric receptor polypeptide is provided,wherein the chimeric receptor polypetide is coded for by a system. Insome alternatives, the system comprises a) a first nucleic acidcomprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte,a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain, and d) apolynucleotide coding for an intracellular signaling domain, and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, thepolypeptide that modulates checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding forchimeric antigen receptor comprising a ligand binding domain, whereinthe ligand is a tumor specific molecule, viral molecule, or any othermolecule expressed on a target cell population, wherein the ligand canelicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor. In some alternatives, thefirst promoter is in opposite orientation to the second promoter. Insome alternatives, the ligand binding domain is an antibody fragment. Insome alternatives, the tumor specific molecule is selected from thegroup consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7,EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR orcombinations thereof. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a cytokine, a chemokine receptor, a polypeptide thatregulates apoptosis, or a polypeptide that modulates checkpointsignaling; and b) a second nucleic acid comprising a second constitutiveor inducible promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the polypeptide that modulates checkpoint signalinginhibits negative checkpoint regulators. In some alternatives, thenegative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3. Insome alternatives, the second nucleic acid further comprises apolynucleotide coding for chimeric antigen receptor comprising a ligandbinding domain, wherein the ligand is a tumor specific molecule, viralmolecule, or any other molecule expressed on a target cell population,wherein the ligand can elicit recognition, modulation, inhibition,and/or elimination by a lymphocyte; a polynucleotide coding for apolypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor; a polynucleotide coding for a transmembranedomain In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment. In some alternatives, the ligandbinding domain is single chain variable fragment. In some alternatives,the tumor specific molecule is selected from the group consisting ofCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. Insome alternatives, the spacer is optimized for increased T cellproliferation and/or cytokine production in response to the ligand ascompared to a reference chimeric receptor. In some alternatives, thesecond promoter is an inducible promoter. In some alternatives, thesecond promoter is constitutive promoter.

In some alternatives, a host cell is provided, wherein the host cellcomprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte, a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain, and d) a polynucleotide coding for an intracellular signalingdomain, and b) a second nucleic acid comprising a second promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the drug is tamoxifenand/or its metabolites. In some alternatives, the first promotercomprises a nucleic acid sequence of SEQ ID NO: 41. In somealternatives, the second promoter is a constitutive promoter. In somealternatives, the second promoter is the EF1αp. In some alternatives,the transcriptional activator comprises a sequence of SEQ ID NO: 40. Insome alternatives, the first nucleic acid further comprises a firstvector and the second nucleic acid further comprises a second vector. Insome alternatives, both vectors are packaged in a viral vector. In somealternatives, the viral vector is a lentivirus. In some alternatives,the first and second nucleic acid comprise a vector. In somealternatives, the first nucleic acid further comprises a nucleic acidsequence coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a nucleic acid coding for aselectable marker. In some alternatives, the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora cytokine, a chemokine receptor, a polypeptide that regulatesapoptosis, or a polypeptide that modulates checkpoint signaling and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the polypeptide that modulatescheckpoint signaling inhibits negative checkpoint regulators. In somealternatives, the negative checkpoint regulator comprises VISTA, LAG-3and/or TIM3. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor. In some alternatives, the first promoter isin opposite orientation to the second promoter. In some alternatives,the ligand binding domain is an antibody fragment. In some alternatives,the tumor specific molecule is selected from the group consisting ofCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. Insome alternatives, the system comprises a) a first nucleic acidcomprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for acytokine, a chemokine receptor, a polypeptide that regulates apoptosis,or a polypeptide that modulates checkpoint signaling; and b) a secondnucleic acid comprising a second constitutive or inducible promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the polypeptide thatmodulates checkpoint signaling inhibits negative checkpoint regulators.In some alternatives, the negative checkpoint regulator comprises VISTA,LAG-3 and/or TIM3. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor; a polynucleotide codingfor a transmembrane domain In some alternatives, the first promoter isin opposite orientation to the second promoter. In some alternatives,the ligand binding domain is an antibody fragment. In some alternatives,the ligand binding domain is single chain variable fragment. In somealternatives, the tumor specific molecule is selected from the groupconsisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR,hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR orcombinations thereof. In some alternatives, the spacer is optimized forincreased T cell proliferation and/or cytokine production in response tothe ligand as compared to a reference chimeric receptor. In somealternatives, the second promoter is an inducible promoter. In somealternatives, the second promoter is constitutive promoter. In somealternatives, the tumor specific molecule is selected from the groupconsisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR,hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR orcombinations thereof. In some alternatives, the host cell is a CD8+ Tcytotoxic lymphocyte cell selected from the group consisting of naïveCD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cellsand bulk CD8+ T cells. In some alternatives, the host cell is a CD4+ Thelper lymphocyte cell that is selected from the group consisting ofnaïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ Tcells, and bulk CD4+ T cells. In some alternatives, the spacer isoptimized for increased T cell proliferation and/or cytokine productionin response to the ligand as compared to a reference chimeric receptor.In some alternatives, the host cell is a precursor T cell. In somealternatives, the precursor T cell is a hematopoietic stem cell.

In some alternatives, a composition is provided, wherein the compositioncomprises a host cell in a pharmaceutically acceptable excipient. Insome alternatives, the host cell comprises a system. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter inducible by a drug, wherein the first nucleic acid isoperably linked to a polynucleotide coding for a chimeric antigenreceptor comprising a ligand binding domain, wherein the ligand bindingdomain is specific for a ligand, wherein the ligand is a tumor specificmolecule, viral molecule, or any other molecule expressed on a targetcell population, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte, a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; apolynucleotide coding for a transmembrane domain, and d) apolynucleotide coding for an intracellular signaling domain, and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, thepolypeptide that modulates checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding forchimeric antigen receptor comprising a ligand binding domain, whereinthe ligand is a tumor specific molecule, viral molecule, or any othermolecule expressed on a target cell population, wherein the ligand canelicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor. In some alternatives, thefirst promoter is in opposite orientation to the second promoter. Insome alternatives, the ligand binding domain is an antibody fragment. Insome alternatives, the tumor specific molecule is selected from thegroup consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7,EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR orcombinations thereof. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a cytokine, a chemokine receptor, a polypeptide thatregulates apoptosis, or a polypeptide that modulates checkpointsignaling; and b) a second nucleic acid comprising a second constitutiveor inducible promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the polypeptide that modulates checkpoint signalinginhibits negative checkpoint regulators. In some alternatives, thenegative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3. Insome alternatives, the second nucleic acid further comprises apolynucleotide coding for chimeric antigen receptor comprising a ligandbinding domain, wherein the ligand is a tumor specific molecule, viralmolecule, or any other molecule expressed on a target cell population,wherein the ligand can elicit recognition, modulation, inhibition,and/or elimination by a lymphocyte; a polynucleotide coding for apolypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor; a polynucleotide coding for a transmembranedomain In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment. In some alternatives, the ligandbinding domain is single chain variable fragment. In some alternatives,the tumor specific molecule is selected from the group consisting ofCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. Insome alternatives, the spacer is optimized for increased T cellproliferation and/or cytokine production in response to the ligand ascompared to a reference chimeric receptor. In some alternatives, thesecond promoter is an inducible promoter. In some alternatives, thesecond promoter is constitutive promoter. In some alternatives, the hostcell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells. In some alternatives, thehost cell is a CD4+ T helper lymphocyte cell that is selected from thegroup consisting of naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells, and bulk CD4+ T cells. In somealternatives, the composition comprises a host cell wherein the hostcell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells and further comprises anotherhost cell wherein the host cell is a CD4+ T helper lymphocyte cell thatis selected from the group consisting of naïve CD4+ T cells, centralmemory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ Tcells. In some alternatives, the composition comprises a host cellwherein the host cell is a CD8+ T cytotoxic lymphocyte cell selectedfrom the group consisting of naïve CD8+ T cells, central memory CD8+ Tcells, effector memory CD8+ T cells and bulk CD8+ T cells or a CD4+ Thelper lymphocyte cell that is selected from the group consisting ofnaïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ Tcells, and bulk CD4+ T cells and a second host cell, wherein the secondhost cell is a precursor T cell. In some alternatives, the precursor Tcell is a hematopoietic stem cell.

In some alternatives, an in vitro method for preparing a host cell isprovided wherein the method comprises a) providing a system and b)introducing the system into a separate isolated T lymphocyte populationand expanding each T lymphocyte population in vitro. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter inducible by a drug, wherein the first nucleic acid isoperably linked to a polynucleotide coding for a chimeric antigenreceptor comprising a ligand binding domain, wherein the ligand bindingdomain is specific for a ligand, wherein the ligand is a tumor specificmolecule, viral molecule, or any other molecule expressed on a targetcell population, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte, a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; apolynucleotide coding for a transmembrane domain, and d) apolynucleotide coding for an intracellular signaling domain, and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, thepolypeptide that modulates checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding forchimeric antigen receptor comprising a ligand binding domain, whereinthe ligand is a tumor specific molecule, viral molecule, or any othermolecule expressed on a target cell population, wherein the ligand canelicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor. In some alternatives, thefirst promoter is in opposite orientation to the second promoter. Insome alternatives, the ligand binding domain is an antibody fragment. Insome alternatives, the tumor specific molecule is selected from thegroup consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7,EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR orcombinations thereof. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a cytokine, a chemokine receptor, a polypeptide thatregulates apoptosis, or a polypeptide that modulates checkpointsignaling; and b) a second nucleic acid comprising a second constitutiveor inducible promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the polypeptide that modulates checkpoint signalinginhibits negative checkpoint regulators. In some alternatives, thenegative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3. Insome alternatives, the second nucleic acid further comprises apolynucleotide coding for chimeric antigen receptor comprising a ligandbinding domain, wherein the ligand is a tumor specific molecule, viralmolecule, or any other molecule expressed on a target cell population,wherein the ligand can elicit recognition, modulation, inhibition,and/or elimination by a lymphocyte; a polynucleotide coding for apolypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor; a polynucleotide coding for a transmembranedomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment. In some alternatives, the ligandbinding domain is single chain variable fragment. In some alternatives,the tumor specific molecule is selected from the group consisting ofCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. Insome alternatives, the spacer is optimized for increased T cellproliferation and/or cytokine production in response to the ligand ascompared to a reference chimeric receptor. In some alternatives, thesecond promoter is an inducible promoter. In some alternatives, thesecond promoter is constitutive promoter. In some alternatives, whereinthe T lymphocytes are expanded, the method further comprises culturingthe cells in the presence of anti-CD3 and/or anti CD28, and at least onehomeostatic cytokine until the cells expand sufficiently for use as acell infusion. In some alternatives, the lymphocyte is CD8+ or CD4+. Insome alternatives, the cells are precursor T cells. In somealternatives, the cells are hematopoietic stem cells. In somealternatives, the composition comprises a host cell wherein the hostcell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells or a CD4+ T helper lymphocytecell that is selected from the group consisting of naïve CD4+ T cells,central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+T cells and a second host cell, wherein the second host cell is aprecursor T cell. In some alternatives, the precursor T cell is ahematopoietic stem cell.

In some alternatives, a use of a host cell or a composition incombination with a drug that induces expression of a transgene in thehost cell or composition for the treatment of cancer or a viralinfection is provided. In some alternatives, the host cell comprises asystem. In some alternatives, the system comprises a) a first nucleicacid comprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for achimeric antigen receptor comprising a ligand binding domain, whereinthe ligand binding domain is specific for a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population, wherein the ligand can elicitrecognition, modulation, inhibition, and/or elimination by a lymphocyte,a polynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain, and d) apolynucleotide coding for an intracellular signaling domain, and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the drug is tamoxifen and/or itsmetabolites. In some alternatives, the first promoter comprises anucleic acid sequence of SEQ ID NO: 41. In some alternatives, the secondpromoter is a constitutive promoter. In some alternatives, the secondpromoter is the EF1αp. In some alternatives, the transcriptionalactivator comprises a sequence of SEQ ID NO: 40. In some alternatives,the first nucleic acid further comprises a first vector and the secondnucleic acid further comprises a second vector. In some alternatives,both vectors are packaged in a viral vector. In some alternatives, theviral vector is a lentivirus. In some alternatives, the first and secondnucleic acid comprise a vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker. In some alternatives, the second nucleic acid further comprisesa nucleic acid coding for a selectable marker. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoterinducible by a drug, wherein the first nucleic acid is operably linkedto a polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, thepolypeptide that modulates checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding forchimeric antigen receptor comprising a ligand binding domain, whereinthe ligand is a tumor specific molecule, viral molecule, or any othermolecule expressed on a target cell population, wherein the ligand canelicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor. In some alternatives, thefirst promoter is in opposite orientation to the second promoter. Insome alternatives, the ligand binding domain is an antibody fragment. Insome alternatives, the tumor specific molecule is selected from thegroup consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7,EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR orcombinations thereof. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a cytokine, a chemokine receptor, a polypeptide thatregulates apoptosis, or a polypeptide that modulates checkpointsignaling; and b) a second nucleic acid comprising a second constitutiveor inducible promoter operably linked to nucleic acid coding for atranscriptional activator for the inducible promoter. In somealternatives, the polypeptide that modulates checkpoint signalinginhibits negative checkpoint regulators. In some alternatives, thenegative checkpoint regulator comprises VISTA, LAG-3 and/or TIM3. Insome alternatives, the second nucleic acid further comprises apolynucleotide coding for chimeric antigen receptor comprising a ligandbinding domain, wherein the ligand is a tumor specific molecule, viralmolecule, or any other molecule expressed on a target cell population,wherein the ligand can elicit recognition, modulation, inhibition,and/or elimination by a lymphocyte; a polynucleotide coding for apolypeptide spacer, wherein the spacer is optimized; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor; a polynucleotide coding for a transmembranedomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment. In some alternatives, the ligandbinding domain is single chain variable fragment. In some alternatives,the tumor specific molecule is selected from the group consisting ofCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. Insome alternatives, the spacer is optimized for increased T cellproliferation and/or cytokine production in response to the ligand ascompared to a reference chimeric receptor. In some alternatives, thesecond promoter is an inducible promoter. In some alternatives, thesecond promoter is constitutive promoter. In some alternatives, the hostcell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells. In some alternatives, thehost cell is a CD4+ T helper lymphocyte cell that is selected from thegroup consisting of naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells, and bulk CD4+ T cells. In somealternatives, the composition comprises a host cell in apharmaceutically acceptable excipient. In some alternatives, the hostcell comprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain; and b) a second nucleic acid comprising a second promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the second promoter isconstitutive or inducible. In some alternatives, the drug is tamoxifenand/or its metabolites. In some alternatives, the first promotercomprises a nucleic acid sequence of SEQ ID NO:41. In some alternatives,the second promoter is a constitutive promoter. In some alternatives,the second promoter is the EF1αp. In some alternatives, thetranscriptional activator comprises a sequence of SEQ ID NO:40. In somealternatives, the first nucleic acid further comprises a first vectorand the second nucleic acid further comprises a second vector. In somealternatives, both vectors are packaged in a viral vector. In somealternatives, the viral vector is a lentivirus. In some alternatives,the first and second nucleic acid comprise a vector. In somealternatives, the first nucleic acid further comprises a nucleic acidsequence coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a nucleic acid coding for aselectable marker. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; apolynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment. In some alternatives, the tumor specific molecule isselected from the group consisting of CD19, CD20, CD22, CD23, CD123,CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, andMAGE A3 TCR or combinations thereof. In some alternatives, the systemcomprises a) a first nucleic acid comprising a first promoter inducibleby a drug, wherein the first nucleic acid is operably linked to apolynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling; and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the secondpromoter is constitutive or inducible. In some alternatives, the secondnucleic acid further comprises a polynucleotide coding for chimericantigen receptor comprising a ligand binding domain, wherein the ligandbinding domain is specific for a ligand, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment. In some alternatives, the ligand binding domain issingle chain variable fragment. In some alternatives, the tumor specificmolecule is selected from the group consisting of CD19, CD20, CD22,CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA,Her2, GD-2, and MAGE A3 TCR or combinations thereof. In somealternatives, the host cell is a precursor T cell. In some alternatives,the precursor T cell is a hematopoietic stem cell. In some alternatives,the isolated T lymphocyte population comprises precursor T cells. Insome alternatives, the precursor T cells are hematopoietic stem cells.In some alternatives, the host cell is a CD8+ T cytotoxic lymphocytecell selected from the group consisting of naïve CD8+ T cells, centralmemory CD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells.In some alternatives, the host cell is a CD4+ T helper lymphocyte cellthat is selected from the group consisting of naïve CD4+ T cells,central memory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+T cells. In some alternatives, the composition comprises a host cellwherein the host cell is a CD8+ T cytotoxic lymphocyte cell selectedfrom the group consisting of naïve CD8+ T cells, central memory CD8+ Tcells, effector memory CD8+ T cells and bulk CD8+ T cells and anotherhost cell wherein the host cell is a CD4+ T helper lymphocyte cell thatis selected from the group consisting of naïve CD4+ T cells, centralmemory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ Tcells. In some alternatives, the cancer is a solid tumor or hematologicmalignancy. In some alternatives, the solid tumor is selected from thegroup consisting of a breast cancer, brain cancer, lung cancer, coloncancer, renal cancer, pancreatic cancer, prostate cancer, and ovariancancer. In some alternatives, the composition comprises a host cellwherein the host cell is a CD8+ T cytotoxic lymphocyte cell selectedfrom the group consisting of naïve CD8+ T cells, central memory CD8+ Tcells, effector memory CD8+ T cells and bulk CD8+ T cells or a CD4+ Thelper lymphocyte cell that is selected from the group consisting ofnaïve CD4+ T cells, central memory CD4+ T cells, effector memory CD4+ Tcells, and bulk CD4+ T cells and a second host cell, wherein the secondhost cell is a precursor T cell. In some alternatives, the precursor Tcell is a hematopoietic stem cell.

In some alternatives, a method of performing cellular immunotherapy in asubject having cancer or a viral infection is provided wherein themethod comprises administering a composition or a host cell to thesubject and administering a drug that induces expression of a transgenein the composition or the host cells. In some alternatives, the hostcell comprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte, a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain, and d) a polynucleotide coding for an intracellular signalingdomain, and b) a second nucleic acid comprising a second promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the drug is tamoxifenand/or its metabolites. In some alternatives, the first promotercomprises a nucleic acid sequence of SEQ ID NO: 41. In somealternatives, the second promoter is a constitutive promoter. In somealternatives, the second promoter is the EF1αp. In some alternatives,the transcriptional activator comprises a sequence of SEQ ID NO: 40. Insome alternatives, the first nucleic acid further comprises a firstvector and the second nucleic acid further comprises a second vector. Insome alternatives, both vectors are packaged in a viral vector. In somealternatives, the viral vector is a lentivirus. In some alternatives,the first and second nucleic acid comprise a vector. In somealternatives, the first nucleic acid further comprises a nucleic acidsequence coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a nucleic acid coding for aselectable marker. In some alternatives, the system comprises a) a firstnucleic acid comprising a first promoter inducible by a drug, whereinthe first nucleic acid is operably linked to a polynucleotide coding fora cytokine, a chemokine receptor, a polypeptide that regulatesapoptosis, or a polypeptide that modulates checkpoint signaling and b) asecond nucleic acid comprising a second promoter operably linked tonucleic acid coding for a transcriptional activator for the induciblepromoter. In some alternatives, the polypeptide that modulatescheckpoint signaling inhibits negative checkpoint regulators. In somealternatives, the negative checkpoint regulator comprises VISTA, LAG-3and/or TIM3. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; a polynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the spacer is optimized for increased T cell proliferationand/or cytokine production in response to the ligand as compared to areference chimeric receptor. In some alternatives, the first promoter isin opposite orientation to the second promoter. In some alternatives,the ligand binding domain is an antibody fragment. In some alternatives,the tumor specific molecule is selected from the group consisting ofCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. Insome alternatives, the system comprises a) a first nucleic acidcomprising a first promoter inducible by a drug, wherein the firstnucleic acid is operably linked to a polynucleotide coding for acytokine, a chemokine receptor, a polypeptide that regulates apoptosis,or a polypeptide that modulates checkpoint signaling; and b) a secondnucleic acid comprising a second constitutive or inducible promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the polypeptide thatmodulates checkpoint signaling inhibits negative checkpoint regulators.In some alternatives, the negative checkpoint regulator comprises VISTA,LAG-3 and/or TIM3. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand is a tumorspecific molecule, viral molecule, or any other molecule expressed on atarget cell population, wherein the ligand can elicit recognition,modulation, inhibition, and/or elimination by a lymphocyte; apolynucleotide coding for a polypeptide spacer, wherein the spacer isoptimized; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the spacer is optimized for increased Tcell proliferation and/or cytokine production in response to the ligandas compared to a reference chimeric receptor; a polynucleotide codingfor a transmembrane domain. In some alternatives, the first promoter isin opposite orientation to the second promoter. In some alternatives,the ligand binding domain is an antibody fragment. In some alternatives,the ligand binding domain is single chain variable fragment. In somealternatives, the tumor specific molecule is selected from the groupconsisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR,hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR orcombinations thereof. In some alternatives, the spacer is optimized forincreased T cell proliferation and/or cytokine production in response tothe ligand as compared to a reference chimeric receptor. In somealternatives, the second promoter is an inducible promoter. In somealternatives, the second promoter is constitutive promoter. In somealternatives, the host cell is a precursor T cell. In some alternatives,the host cell is a hematopoietic stem cell. In some alternatives, thehost cell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cells. In some alternatives, thehost cell is a CD4+ T helper lymphocyte cell that is selected from thegroup consisting of naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells, and bulk CD4+ T cells. In somealternatives, the composition comprises a host cell in apharmaceutically acceptable excipient. In some alternatives, the hostcell comprises a system. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter inducible by a drug,wherein the first nucleic acid is operably linked to a polynucleotidecoding for a chimeric antigen receptor comprising a ligand bindingdomain, wherein the ligand binding domain is specific for a ligand,wherein the ligand is a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population, wherein the ligandcan elicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain; and b) a second nucleic acid comprising a second promoteroperably linked to nucleic acid coding for a transcriptional activatorfor the inducible promoter. In some alternatives, the second promoter isconstitutive or inducible. In some alternatives, the drug is tamoxifenand/or its metabolites. In some alternatives, the first promotercomprises a nucleic acid sequence of SEQ ID NO: 41. In somealternatives, the second promoter is a constitutive promoter. In somealternatives, the second promoter is the EF1αp. In some alternatives,the transcriptional activator comprises a sequence of SEQ ID NO: 40. Insome alternatives, the first nucleic acid further comprises a firstvector and the second nucleic acid further comprises a second vector. Insome alternatives, both vectors are packaged in a viral vector. In somealternatives, the viral vector is a lentivirus. In some alternatives,the first and second nucleic acid comprise a vector. In somealternatives, the first nucleic acid further comprises a nucleic acidsequence coding for a selectable marker. In some alternatives, thesecond nucleic acid further comprises a nucleic acid coding for aselectable marker. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for chimeric antigen receptorcomprising a ligand binding domain, wherein the ligand binding domain isspecific for a ligand, wherein the ligand is a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation, wherein the ligand can elicit recognition, modulation,inhibition, and/or elimination by a lymphocyte; a polynucleotide codingfor a polypeptide spacer, wherein the spacer is optimized; apolynucleotide coding for a transmembrane domain; and d) apolynucleotide coding for an intracellular signaling domain. In somealternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment. In some alternatives, the tumor specific molecule isselected from the group consisting of CD19, CD20, CD22, CD23, CD123,CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, andMAGE A3 TCR or combinations thereof. In some alternatives, the systemcomprises a) a first nucleic acid comprising a first promoter inducibleby a drug, wherein the first nucleic acid is operably linked to apolynucleotide coding for a cytokine, a chemokine receptor, apolypeptide that regulates apoptosis, or a polypeptide that modulatescheckpoint signaling; and b) a second nucleic acid comprising a secondpromoter operably linked to nucleic acid coding for a transcriptionalactivator for the inducible promoter. In some alternatives, the secondpromoter is constitutive or inducible. In some alternatives, thepolypeptide that modulates checkpoint signaling inhibits negativecheckpoint regulators. In some alternatives, the negative checkpointregulator comprises VISTA, LAG-3 and/or TIM3. In some alternatives, thesecond nucleic acid further comprises a polynucleotide coding forchimeric antigen receptor comprising a ligand binding domain, whereinthe ligand is a tumor specific molecule, viral molecule, or any othermolecule expressed on a target cell population, wherein the ligand canelicit recognition, modulation, inhibition, and/or elimination by alymphocyte; a polynucleotide coding for a polypeptide spacer, whereinthe spacer is optimized; a polynucleotide coding for a transmembranedomain; and d) a polynucleotide coding for an intracellular signalingdomain. In some alternatives, the first promoter is in oppositeorientation to the second promoter. In some alternatives, the ligandbinding domain is an antibody fragment. In some alternatives, the ligandbinding domain is single chain variable fragment. In some alternatives,the tumor specific molecule is selected from the group consisting ofCD19, CD20, CD22, CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin,c-Met, PSMA, Her2, GD-2, and MAGE A3 TCR or combinations thereof. Insome alternatives, the host cell is a precursor T cell. In somealternatives, the host cell is a hematopoietic stem cell. In somealternatives, the host cell is a CD8+ T cytotoxic lymphocyte cellselected from the group consisting of naïve CD8+ T cells, central memoryCD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cells. Insome alternatives, the host cell is a CD4+ T helper lymphocyte cell thatis selected from the group consisting of naïve CD4+ T cells, centralmemory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ Tcells. In some alternatives, the composition comprises a host cellwherein the host cell is a CD8+ T cytotoxic lymphocyte cell selectedfrom the group consisting of naïve CD8+ T cells, central memory CD8+ Tcells, effector memory CD8+ T cells and bulk CD8+ T cells and anotherhost cell wherein the host cell is a CD4+ T helper lymphocyte cell thatis selected from the group consisting of naïve CD4+ T cells, centralmemory CD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ Tcells. In some alternatives, the cancer is selected from a solid tumoror hematologic malignancy. In some alternatives, the solid tumor isselected from the group consisting of a breast cancer, brain cancer,lung cancer, colon cancer, renal cancer, pancreatic cancer, prostatecancer, and ovarian cancer. In some alternatives, the host cell is aprecursor T cell. In some alternatives, the precursor T cell is ahematopoietic stem cell. In some alternatives, the isolated T lymphocytepopulation comprises precursor T cells. In some alternatives, theprecursor T cells are hematopoietic stem cells. In some alternatives,the composition comprises a host cell wherein the host cell is a CD8+ Tcytotoxic lymphocyte cell selected from the group consisting of naïveCD8+ T cells, central memory CD8+ T cells, effector memory CD8+ T cellsand bulk CD8+ T cells or a CD4+ T helper lymphocyte cell that isselected from the group consisting of naïve CD4+ T cells, central memoryCD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells and asecond host cell, wherein the second host cell is a precursor T cell. Insome alternatives, the precursor T cell is a hematopoietic stem cell.

More Alternatives.

In some alternatives, a system for inducible expression of a chimericantigen receptor, the system is provided, wherein the system comprisesa) a first nucleic acid comprising a first promoter, which is aninducible promoter, operably linked to a polynucleotide coding for achimeric antigen receptor and b) a second nucleic acid comprising asecond promoter operably linked to a polynucleotide coding for atranscriptional activator, which is capable of activating transcriptionfrom the first promoter in the presence of a drug or metabolite thereof.In some alternatives, a system for inducible expression is provided,wherein the system comprises a) an inducible promoter, b) apolynucleotide coding for a chimeric antigen receptor and c) apolynucleotide coding for a transcriptional activator, whichtranscriptional activator is capable of activating transcription fromthe inducible promoter in the presence of a drug or metabolite thereof.In some alternatives, the polynucleotide coding for the chimericreceptor is operably linked to the inducible promoter. In somealternatives, the system further comprises further comprising apolynucleotide encoding a recombinant protein, which polynucleotide isoperably linked to the inducible promoter. In some alternatives, thedrug or metabolite thereof comprises: (i) a drug tolerated whenadministered to a human subject daily or weekly, or a metabolitethereof; (ii) a molecule that specifically binds to a human receptor,optionally the estrogen receptor, or a metabolite thereof; and/or (iii)tamoxifen and/or a metabolite or analog of tamoxifen. In somealternatives, the transcriptional activator comprises: (a) a DNA-bindingdomain; (b) a ligand-binding domain that specifically binds to the drugor metabolite thereof; and (c) a transactivation domain, optionallylinked and/or fused in that order. In some alternatives of the system,(a) the DNA-binding domain comprises DNA binding sites not present in aprotein naturally expressed in a lymphocyte or not present in a proteinnaturally expressed in a T cell; and/or (b) the drug or metabolite isthe molecule that specifically binds to the human receptor, optionallyestrogen receptor, or metabolite thereof, and the binding between thedrug or metabolite and the ligand-binding domain is selective for theligand-binding domain over the human receptor, whereby binding by theligand-binding domain to the drug or metabolite is greater, optionallyat least 1.5, 2, 3, or 4 times as strong, as the binding by the humanreceptor; and/or (c) the transactivation domain comprises a p65transactivation domain or functional variant thereof; and/or (d) thefirst promoter comprises one or more binding sites for the DNA bindingdomain. In some alternatives, the first promoter does not compriseanother binding site for any human DNA binding domain other than aDNA-binding domain or domains present in the transcriptional activator;and/or wherein the first promoter is a synthetic chimeric promoterand/or the transcriptional activator is a synthetic chimerictranscriptional activator; and/or wherein the DNA binding domaincomprises a DNA binding domain present in a hepatocyte nuclear factor,which is optionally HNF1-alpha or HNF1-beta. In some alternatives of thesystem, the first promoter comprises the nucleic acid sequence of SEQ IDNO: 41. In some alternatives, the second promoter is a constitutivepromoter. In some alternatives, the second promoter is or comprises anEF1α promoter or functional portion thereof. In some alternatives, thetranscriptional activator comprises a polypeptide having the sequence ofSEQ ID NO: 40. In some alternatives, the first nucleic acid is comprisedwithin a first vector, which is further comprised by the system and thesecond nucleic acid is comprised within a second vector, which isfurther comprised by the system. In some alternatives, the first nucleicacid and the second nucleic acid or the first promoter, polynucleotideencoding the chimeric antigen receptor, second promoter, andpolynucleotide encoding the transactivator, are comprised within avector, which is further comprised by the system. In some alternativesof the system, the system is comprised in a single viral packagingvector. In some alternatives, the viral vector is a lentiviral vector.In some alternatives, the first nucleic acid further comprises a nucleicacid sequence coding for a selectable marker or wherein the systemfurther comprises a selectable marker operably linked to the firstpromoter. In some alternatives, the second nucleic acid furthercomprises a nucleic acid coding for a selectable marker or wherein thesystem further comprises a selectable marker operably linked to thesecond promoter.

In some alternatives, a system for inducible expression is provided,wherein the system comprises a) a first nucleic acid comprising a firstpromoter, which is an inducible promoter and is operably linked to apolynucleotide coding for a cytokine, a chemokine receptor, apolypeptide than inhibits apoptosis, or a polypeptide that inhibitsnegative checkpoint signaling and b) a second nucleic acid comprising asecond promoter, which is a constitutive or inducible promoter, operablylinked to a polynucleotide coding for a transcriptional activatorcapable of inducing transcription from the first promoter in thepresence of a drug or metabolite or analog thereof. In somealternatives, the second nucleic acid further comprises a polynucleotidecoding for a recombinant antigen receptor, which optionally is achimeric antigen receptor. In some alternatives, the chimeric antigenreceptor comprises a a) ligand binding domain, which binds to a ligandthat is optionally a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population that is suitable tomediate recognition and elimination by a lymphocyte b) a polypeptidespacer, wherein the spacer optionally provides for increased T cellproliferation and/or cytokine production in response to the ligand ascompared to a reference chimeric receptor, c) a transmembrane domain andd) an intracellular signaling domain. In some alternatives, the firstpromoter is in opposite orientation to the second promoter. In somealternatives, the ligand binding domain is an antibody fragment. In somealternatives, the ligand binding domain is single chain variablefragment. In some alternatives, the tumor specific molecule is selectedfrom the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1,CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, MAGE A3 TCR andcombinations thereof.

In some alternatives, a chimeric receptor polypeptide encoded by thesystem is provided. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter, which is an induciblepromoter, operably linked to a polynucleotide coding for a chimericantigen receptor and b) a second nucleic acid comprising a secondpromoter operably linked to a polynucleotide coding for atranscriptional activator, which is capable of activating transcriptionfrom the first promoter in the presence of a drug or metabolite thereof.In some alternatives, a system for inducible expression is provided,wherein the system comprises a) an inducible promoter, b) apolynucleotide coding for a chimeric antigen receptor and c) apolynucleotide coding for a transcriptional activator, whichtranscriptional activator is capable of activating transcription fromthe inducible promoter in the presence of a drug or metabolite thereof.In some alternatives, the polynucleotide coding for the chimericreceptor is operably linked to the inducible promoter. In somealternatives, the system further comprises further comprising apolynucleotide encoding a recombinant protein, which polynucleotide isoperably linked to the inducible promoter. In some alternatives, thedrug or metabolite thereof comprises: (i) a drug tolerated whenadministered to a human subject daily or weekly, or a metabolitethereof; (ii) a molecule that specifically binds to a human receptor,optionally the estrogen receptor, or a metabolite thereof; and/or(iii)tamoxifen and/or a metabolite or analog of tamoxifen. In somealternatives, the transcriptional activator comprises: (a) a DNA-bindingdomain; (b) a ligand-binding domain that specifically binds to the drugor metabolite thereof; and (c) a transactivation domain, optionallylinked and/or fused in that order. In some alternatives of the system,(a) the DNA-binding domain comprises DNA binding sites not present in aprotein naturally expressed in a lymphocyte or not present in a proteinnaturally expressed in a T cell; and/or (b) the drug or metabolite isthe molecule that specifically binds to the human receptor, optionallyestrogen receptor, or metabolite thereof, and the binding between thedrug or metabolite and the ligand-binding domain is selective for theligand-binding domain over the human receptor, whereby binding by theligand-binding domain to the drug or metabolite is greater, optionallyat least 1.5, 2, 3, or 4 times as strong, as the binding by the humanreceptor; and/or (c) the transactivation domain comprises a p65transactivation domain or functional variant thereof; and/or (d) thefirst promoter comprises one or more binding sites for the DNA bindingdomain. In some alternatives, the first promoter does not compriseanother binding site for any human DNA binding domain other than aDNA-binding domain or domains present in the transcriptional activator;and/or wherein the first promoter is a synthetic chimeric promoterand/or the transcriptional activator is a synthetic chimerictranscriptional activator; and/or wherein the DNA binding domaincomprises a DNA binding domain present in a hepatocyte nuclear factor,which is optionally HNF1-alpha or HNF1-beta. The system of any of claims1-8, wherein the first promoter comprises the nucleic acid sequence ofSEQ ID NO: 41. In some alternatives, the second promoter is aconstitutive promoter. In some alternatives, the second promoter is orcomprises an EF1α promoter or functional portion thereof. In somealternatives, the transcriptional activator comprises a polypeptidehaving the sequence of SEQ ID NO: 40. In some alternatives, the firstnucleic acid is comprised within a first vector, which is furthercomprised by the system and the second nucleic acid is comprised withina second vector, which is further comprised by the system. In somealternatives, the first nucleic acid and the second nucleic acid or thefirst promoter, polynucleotide encoding the chimeric antigen receptor,second promoter, and polynucleotide encoding the transactivator, arecomprised within a vector, which is further comprised by the system. Insome alternatives of the system, the system is comprised in a singleviral packaging vector. In some alternatives, the viral vector is alentiviral vector. In some alternatives, the first nucleic acid furthercomprises a nucleic acid sequence coding for a selectable marker orwherein the system further comprises a selectable marker operably linkedto the first promoter. In some alternatives, the second nucleic acidfurther comprises a nucleic acid coding for a selectable marker orwherein the system further comprises a selectable marker operably linkedto the second promoter. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter, which is an induciblepromoter and is operably linked to a polynucleotide coding for acytokine, a chemokine receptor, a polypeptide than inhibits apoptosis,or a polypeptide that inhibits negative checkpoint signaling and b) asecond nucleic acid comprising a second promoter, which is aconstitutive or inducible promoter, operably linked to a polynucleotidecoding for a transcriptional activator capable of inducing transcriptionfrom the first promoter in the presence of a drug or metabolite oranalog thereof. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for a recombinant antigen receptor,which optionally is a chimeric antigen receptor. In some alternatives,the chimeric antigen receptor comprises a a) ligand binding domain,which binds to a ligand that is optionally a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation that is suitable to mediate recognition and elimination by alymphocyte b) a polypeptide spacer, wherein the spacer optionallyprovides for increased T cell proliferation and/or cytokine productionin response to the ligand as compared to a reference chimeric receptor,c) a transmembrane domain and d) an intracellular signaling domain. Insome alternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment. In some alternatives, the ligand binding domain issingle chain variable fragment. In some alternatives, the tumor specificmolecule is selected from the group consisting of CD19, CD20, CD22,CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA,Her2, GD-2, and MAGE A3 TCR and combinations thereof.

In some alternatives, a host cell comprising a system or viral packagingvector is provided. In some alternatives, the system comprises a) afirst nucleic acid comprising a first promoter, which is an induciblepromoter, operably linked to a polynucleotide coding for a chimericantigen receptor and b) a second nucleic acid comprising a secondpromoter operably linked to a polynucleotide coding for atranscriptional activator, which is capable of activating transcriptionfrom the first promoter in the presence of a drug or metabolite thereof.In some alternatives, a system for inducible expression is provided,wherein the system comprises a) an inducible promoter, b) apolynucleotide coding for a chimeric antigen receptor and c) apolynucleotide coding for a transcriptional activator, whichtranscriptional activator is capable of activating transcription fromthe inducible promoter in the presence of a drug or metabolite thereof.In some alternatives, the polynucleotide coding for the chimericreceptor is operably linked to the inducible promoter. In somealternatives, the system further comprises further comprising apolynucleotide encoding a recombinant protein, which polynucleotide isoperably linked to the inducible promoter. In some alternatives, thedrug or metabolite thereof comprises: (i) a drug tolerated whenadministered to a human subject daily or weekly, or a metabolitethereof; (ii) a molecule that specifically binds to a human receptor,optionally the estrogen receptor, or a metabolite thereof; and/or (iii)tamoxifen and/or a metabolite or analog of tamoxifen. In somealternatives, the transcriptional activator comprises: (a) a DNA-bindingdomain; (b) a ligand-binding domain that specifically binds to the drugor metabolite thereof; and (c) a transactivation domain, optionallylinked and/or fused in that order. In some alternatives of the system,(a) the DNA-binding domain comprises DNA binding sites not present in aprotein naturally expressed in a lymphocyte or not present in a proteinnaturally expressed in a T cell; and/or (b) the drug or metabolite isthe molecule that specifically binds to the human receptor, optionallyestrogen receptor, or metabolite thereof, and the binding between thedrug or metabolite and the ligand-binding domain is selective for theligand-binding domain over the human receptor, whereby binding by theligand-binding domain to the drug or metabolite is greater, optionallyat least 1.5, 2, 3, or 4 times as strong, as the binding by the humanreceptor; and/or (c) the transactivation domain comprises a p65transactivation domain or functional variant thereof; and/or (d) thefirst promoter comprises one or more binding sites for the DNA bindingdomain. In some alternatives, the first promoter does not compriseanother binding site for any human DNA binding domain other than aDNA-binding domain or domains present in the transcriptional activator;and/or wherein the first promoter is a synthetic chimeric promoterand/or the transcriptional activator is a synthetic chimerictranscriptional activator; and/or wherein the DNA binding domaincomprises a DNA binding domain present in a hepatocyte nuclear factor,which is optionally HNF1-alpha or HNF1-beta. In some alternatives, thefirst promoter comprises the nucleic acid sequence of SEQ ID NO: 41. Insome alternatives, the second promoter is a constitutive promoter. Insome alternatives, the second promoter is or comprises an EFla promoteror functional portion thereof. In some alternatives, the transcriptionalactivator comprises a polypeptide having the sequence of SEQ ID NO: 40.In some alternatives, the first nucleic acid is comprised within a firstvector, which is further comprised by the system and the second nucleicacid is comprised within a second vector, which is further comprised bythe system. In some alternatives, the first nucleic acid and the secondnucleic acid or the first promoter, polynucleotide encoding the chimericantigen receptor, second promoter, and polynucleotide encoding thetransactivator, are comprised within a vector, which is furthercomprised by the system. In some alternatives, the system describedabove is comprised in a single viral packaging vector. In some suchalternatives, viral vector is a lentiviral vector. In some alternatives,the first nucleic acid further comprises a nucleic acid sequence codingfor a selectable marker or wherein the system further comprises aselectable marker operably linked to the first promoter. In somealternatives, the second nucleic acid further comprises a nucleic acidcoding for a selectable marker or wherein the system further comprises aselectable marker operably linked to the second promoter. In somealternatives, the system comprises a) a first nucleic acid comprising afirst promoter, which is an inducible promoter and is operably linked toa polynucleotide coding for a cytokine, a chemokine receptor, apolypeptide than inhibits apoptosis, or a polypeptide that inhibitsnegative checkpoint signaling and b) a second nucleic acid comprising asecond promoter, which is a constitutive or inducible promoter, operablylinked to a polynucleotide coding for a transcriptional activatorcapable of inducing transcription from the first promoter in thepresence of a drug or metabolite or analog thereof. In somealternatives, the second nucleic acid further comprises a polynucleotidecoding for a recombinant antigen receptor, which optionally is achimeric antigen receptor. In some alternatives, the chimeric antigenreceptor comprises a a) ligand binding domain, which binds to a ligandthat is optionally a tumor specific molecule, viral molecule, or anyother molecule expressed on a target cell population that is suitable tomediate recognition and elimination by a lymphocyte b) a polypeptidespacer, wherein the spacer optionally provides for increased T cellproliferation and/or cytokine production in response to the ligand ascompared to a reference chimeric receptor, c) a transmembrane domain andd) an intracellular signaling domain. In some alternatives, the firstpromoter is in opposite orientation to the second promoter. In somealternatives, the ligand binding domain is an antibody fragment. In somealternatives, the ligand binding domain is single chain variablefragment. In some alternatives, the tumor specific molecule is selectedfrom the group consisting of CD19, CD20, CD22, CD23, CD123, CS-1, ROR1,CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA, Her2, GD-2, and MAGE A3 TCRand combinations thereof.

In some alternatives, a host cell is provided, wherein the host cell isa primary human lymphocyte, wherein the host cell comprises a) a nucleicacid comprising a first promoter, which is an inducible syntheticpromoter containing a binding site for a DNA binding domain notnaturally present in the primary human lymphocyte and b) apolynucleotide coding for a transcriptional activator, thetranscriptional activator comprising i) the DNA binding domain, whereinthe DNA binding domain does not specifically bind to a DNA sequencenaturally present in the primary human lymphocyte; ii) a domain thatspecifically binds to a drug or metabolite thereof and does not bind ordoes not bind with as great a degree of affinity to any moleculenaturally present in the primary human lymphocyte and iii) atransactivation domain, wherein the transcriptional activator is capableof inducing transcription from the first promoter in the presence of thedrug or metabolite thereof and/or upon binding of the drug or metabolitethereof to the domain in (ii). In some alternatives, the cell furthercomprises a polynucleotide encoding a chimeric antigen receptor, whichoptionally is operably linked to the first promoter. In somealternatives, In some alternatives, the cell further comprises a secondpromoter, which is operably linked to the polynucleotide encoding thetranscriptional activator, wherein the second promoter is optionallyconstitutive. In some alternatives, the host cell is a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cell. In some alternatives, the host cell is a CD4+ T helperlymphocyte cell that is selected from the group consisting of naïve CD4+T cells, central memory CD4+ T cells, effector memory CD4+ T cells, andbulk CD4+ T cells.

In some alternatives, a composition is provided, wherein the compositioncomprises a host cell in a pharmaceutically acceptable excipient. Insome alternatives, the host cell the host cell is a primary humanlymphocyte, wherein the host cell comprises a) a nucleic acid comprisinga first promoter, which is an inducible synthetic promoter containing abinding site for a DNA binding domain not naturally present in theprimary human lymphocyte and b) a polynucleotide coding for atranscriptional activator, the transcriptional activator comprising i)the DNA binding domain, wherein the DNA binding domain does notspecifically bind to a DNA sequence naturally present in the primaryhuman lymphocyte; ii) a domain that specifically binds to a drug ormetabolite thereof and does not bind or does not bind with as great adegree of affinity to any molecule naturally present in the primaryhuman lymphocyte and iii) a transactivation domain, wherein thetranscriptional activator is capable of inducing transcription from thefirst promoter in the presence of the drug or metabolite thereof and/orupon binding of the drug or metabolite thereof to the domain in (ii). Insome alternatives, the cell further comprises a polynucleotide encodinga chimeric antigen receptor, which optionally is operably linked to thefirst promoter. In some alternatives, In some alternatives, the cellfurther comprises a second promoter, which is operably linked to thepolynucleotide encoding the transcriptional activator, wherein thesecond promoter is optionally constitutive. In some alternatives, thehost cell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cell. In some alternatives, the hostcell is a CD4+ T helper lymphocyte cell that is selected from the groupconsisting of naïve CD4+ T cells, central memory CD4+ T cells, effectormemory CD4+ T cells, and bulk CD4+ T cells. In some alternatives, thecomposition comprises a CD8+ T cytotoxic lymphocyte cell selected fromthe group consisting of naïve CD8+ T cells, central memory CD8+ T cells,effector memory CD8+ T cells and bulk CD8+ T cell and a CD4+ T helperlymphocyte cell that is selected from the group consisting of naïve CD4+T cells, central memory CD4+ T cells, effector memory CD4+ T cells, andbulk CD4+ T cells.

In some alternatives, an in vitro method for preparing a host cell isprovided and comprises introducing a system into a separate isolated Tlymphocyte population and expanding each T lymphocyte population invitro. In some alternatives, the host cell the host cell is a primaryhuman lymphocyte, wherein the host cell comprises a) a nucleic acidcomprising a first promoter, which is an inducible synthetic promotercontaining a binding site for a DNA binding domain not naturally presentin the primary human lymphocyte and b) a polynucleotide coding for atranscriptional activator, the transcriptional activator comprising i)the DNA binding domain, wherein the DNA binding domain does notspecifically bind to a DNA sequence naturally present in the primaryhuman lymphocyte; ii) a domain that specifically binds to a drug ormetabolite thereof and does not bind or does not bind with as great adegree of affinity to any molecule naturally present in the primaryhuman lymphocyte and iii) a transactivation domain, wherein thetranscriptional activator is capable of inducing transcription from thefirst promoter in the presence of the drug or metabolite thereof and/orupon binding of the drug or metabolite thereof to the domain in (ii). Insome alternatives, the cell further comprises a polynucleotide encodinga chimeric antigen receptor, which optionally is operably linked to thefirst promoter. In some alternatives, In some alternatives, the cellfurther comprises a second promoter, which is operably linked to thepolynucleotide encoding the transcriptional activator, wherein thesecond promoter is optionally constitutive. In some alternatives, thehost cell is a CD8+ T cytotoxic lymphocyte cell selected from the groupconsisting of naïve CD8+ T cells, central memory CD8+ T cells, effectormemory CD8+ T cells and bulk CD8+ T cell. In some alternatives, the hostcell is a CD4+ T helper lymphocyte cell that is selected from the groupconsisting of naïve CD4+ T cells, central memory CD4+ T cells, effectormemory CD4+ T cells, and bulk CD4+ T cells. In some alternatives, thesystem comprises a) a first nucleic acid comprising a first promoter,which is an inducible promoter, operably linked to a polynucleotidecoding for a chimeric antigen receptor and b) a second nucleic acidcomprising a second promoter operably linked to a polynucleotide codingfor a transcriptional activator, which is capable of activatingtranscription from the first promoter in the presence of a drug ormetabolite thereof. In some alternatives, a system for inducibleexpression is provided, wherein the system comprises a) an induciblepromoter, b) a polynucleotide coding for a chimeric antigen receptor andc) a polynucleotide coding for a transcriptional activator, whichtranscriptional activator is capable of activating transcription fromthe inducible promoter in the presence of a drug or metabolite thereof.In some alternatives, the polynucleotide coding for the chimericreceptor is operably linked to the inducible promoter. In somealternatives, the system further comprises further comprising apolynucleotide encoding a recombinant protein, which polynucleotide isoperably linked to the inducible promoter. In some alternatives, thedrug or metabolite thereof comprises: (i) a drug tolerated whenadministered to a human subject daily or weekly, or a metabolitethereof; (ii) a molecule that specifically binds to a human receptor,optionally the estrogen receptor, or a metabolite thereof; and/or (iii)tamoxifen and/or a metabolite or analog of tamoxifen. In somealternatives, the transcriptional activator comprises: (a) a DNA-bindingdomain; (b) a ligand-binding domain that specifically binds to the drugor metabolite thereof; and (c) a transactivation domain, optionallylinked and/or fused in that order. In some alternatives of the system,(a) the DNA-binding domain comprises DNA binding sites not present in aprotein naturally expressed in a lymphocyte or not present in a proteinnaturally expressed in a T cell; and/or (b) the drug or metabolite isthe molecule that specifically binds to the human receptor, optionallyestrogen receptor, or metabolite thereof, and the binding between thedrug or metabolite and the ligand-binding domain is selective for theligand-binding domain over the human receptor, whereby binding by theligand-binding domain to the drug or metabolite is greater, optionallyat least 1.5, 2, 3, or 4 times as strong, as the binding by the humanreceptor; and/or (c) the transactivation domain comprises a p65transactivation domain or functional variant thereof; and/or (d) thefirst promoter comprises one or more binding sites for the DNA bindingdomain. In some alternatives, the first promoter does not compriseanother binding site for any human DNA binding domain other than aDNA-binding domain or domains present in the transcriptional activator;and/or wherein the first promoter is a synthetic chimeric promoterand/or the transcriptional activator is a synthetic chimerictranscriptional activator; and/or wherein the DNA binding domaincomprises a DNA binding domain present in a hepatocyte nuclear factor,which is optionally HNF1-alpha or HNF1-beta. In some alternatives, thefirst promoter comprises the nucleic acid sequence of SEQ ID NO: 41. Insome alternatives, the second promoter is a constitutive promoter. Insome alternatives, the second promoter is or comprises an EF1α promoteror functional portion thereof. In some alternatives, the transcriptionalactivator comprises a polypeptide having the sequence of SEQ ID NO: 40.In some alternatives, the first nucleic acid is comprised within a firstvector, which is further comprised by the system and the second nucleicacid is comprised within a second vector, which is further comprised bythe system. In some alternatives, the first nucleic acid and the secondnucleic acid or the first promoter, polynucleotide encoding the chimericantigen receptor, second promoter, and polynucleotide encoding thetransactivator, are comprised within a vector, which is furthercomprised by the system. In some alternatives, the system is comprisedin a single viral packaging vector. In some alternatives, the viralvector is a lentiviral vector. In some alternatives, the first nucleicacid further comprises a nucleic acid sequence coding for a selectablemarker or wherein the system further comprises a selectable markeroperably linked to the first promoter. In some alternatives, the secondnucleic acid further comprises a nucleic acid coding for a selectablemarker or wherein the system further comprises a selectable markeroperably linked to the second promoter. In some alternatives, the systemcomprises a) a first nucleic acid comprising a first promoter, which isan inducible promoter and is operably linked to a polynucleotide codingfor a cytokine, a chemokine receptor, a polypeptide than inhibitsapoptosis, or a polypeptide that inhibits negative checkpoint signalingand b) a second nucleic acid comprising a second promoter, which is aconstitutive or inducible promoter, operably linked to a polynucleotidecoding for a transcriptional activator capable of inducing transcriptionfrom the first promoter in the presence of a drug or metabolite oranalog thereof. In some alternatives, the second nucleic acid furthercomprises a polynucleotide coding for a recombinant antigen receptor,which optionally is a chimeric antigen receptor. In some alternatives,the chimeric antigen receptor comprises a a) ligand binding domain,which binds to a ligand that is optionally a tumor specific molecule,viral molecule, or any other molecule expressed on a target cellpopulation that is suitable to mediate recognition and elimination by alymphocyte b) a polypeptide spacer, wherein the spacer optionallyprovides for increased T cell proliferation and/or cytokine productionin response to the ligand as compared to a reference chimeric receptor,c) a transmembrane domain and d) an intracellular signaling domain. Insome alternatives, the first promoter is in opposite orientation to thesecond promoter. In some alternatives, the ligand binding domain is anantibody fragment. In some alternatives, the ligand binding domain issingle chain variable fragment. In some alternatives, the tumor specificmolecule is selected from the group consisting of CD19, CD20, CD22,CD23, CD123, CS-1, ROR1, CE7, EGFR, hB7H3, mesothelin, c-Met, PSMA,Her2, GD-2, MAGE A3 TCR and combinations thereof. In some alternatives,the T lymphocytes in the population are expanded and wherein the methodfurther comprises culturing the cells in the presence of anti-CD3 and/oranti CD28, and at least one homeostatic cytokine until the cells expandsufficiently for use as a cell infusion. In some alternatives, culturingin the presence of anti-CD3 and/or anti CD28, and at least onehomeostatic cytokine can be performed before or after the introductionof the system.

In some alternatives, a use of the host cell or a composition incombination with the drug or a metabolite thereof for the treatment ofcancer or a viral infection is provided. In some alternatives, the hostcell is a primary human lymphocyte, wherein the host cell comprises a) anucleic acid comprising a first promoter, which is an induciblesynthetic promoter containing a binding site for a DNA binding domainnot naturally present in the primary human lymphocyte and b) apolynucleotide coding for a transcriptional activator, thetranscriptional activator comprising i) the DNA binding domain, whereinthe DNA binding domain does not specifically bind to a DNA sequencenaturally present in the primary human lymphocyte; ii) a domain thatspecifically binds to a drug or metabolite thereof and does not bind ordoes not bind with as great a degree of affinity to any moleculenaturally present in the primary human lymphocyte and iii) atransactivation domain, wherein the transcriptional activator is capableof inducing transcription from the first promoter in the presence of thedrug or metabolite thereof and/or upon binding of the drug or metabolitethereof to the domain in (ii). In some alternatives, the cell furthercomprises a polynucleotide encoding a chimeric antigen receptor, whichoptionally is operably linked to the first promoter. In somealternatives, In some alternatives, the cell further comprises a secondpromoter, which is operably linked to the polynucleotide encoding thetranscriptional activator, wherein the second promoter is optionallyconstitutive. In some alternatives, the host cell is a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cell. In some alternatives, the host cell is a CD4+ T helperlymphocyte cell that is selected from the group consisting of naïve CD4+T cells, central memory CD4+ T cells, effector memory CD4+ T cells, andbulk CD4+ T cells. In some alternatives, the composition comprises ahost cell in a pharmaceutically acceptable excipient. In somealternatives, the host cell the host cell is a primary human lymphocyte,wherein the host cell comprises a) a nucleic acid comprising a firstpromoter, which is an inducible synthetic promoter containing a bindingsite for a DNA binding domain not naturally present in the primary humanlymphocyte and b) a polynucleotide coding for a transcriptionalactivator, the transcriptional activator comprising i) the DNA bindingdomain, wherein the DNA binding domain does not specifically bind to aDNA sequence naturally present in the primary human lymphocyte; ii) adomain that specifically binds to a drug or metabolite thereof and doesnot bind or does not bind with as great a degree of affinity to anymolecule naturally present in the primary human lymphocyte and iii) atransactivation domain, wherein the transcriptional activator is capableof inducing transcription from the first promoter in the presence of thedrug or metabolite thereof and/or upon binding of the drug or metabolitethereof to the domain in (ii). In some alternatives, the cell furthercomprises a polynucleotide encoding a chimeric antigen receptor, whichoptionally is operably linked to the first promoter. In somealternatives, the cell further comprises a second promoter, which isoperably linked to the polynucleotide encoding the transcriptionalactivator, wherein the second promoter is optionally constitutive. Insome alternatives, the host cell is a CD8+ T cytotoxic lymphocyte cellselected from the group consisting of naïve CD8+ T cells, central memoryCD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cell. In somealternatives, the host cell is a CD4+ T helper lymphocyte cell that isselected from the group consisting of naïve CD4+ T cells, central memoryCD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. Insome alternatives, the composition comprises a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cell and a CD4+ T helper lymphocyte cell that is selectedfrom the group consisting of naïve CD4+ T cells, central memory CD4+ Tcells, effector memory CD4+ T cells, and bulk CD4+ T cells.

In some alternatives, a cell and a drug for use in treatment orinhibition of cancer or a viral infection is provided, wherein the cellcomprises: (a) a polynucleotide encoding a chimeric antigen receptorthat specifically binds to an antigen associated with the cancer or theviral infection, (b) an inducible synthetic promoter, and (c) atranscriptional activator containing a DNA binding domain thatspecifically binds to the synthetic promoter and a domain thatspecifically binds to the drug or a metabolite thereof and is capable ofinducing transcription from the synthetic promoter in the presence ofthe drug or a metabolite thereof. In some alternatives, the cancer is asolid tumor or hematologic malignancy. In some alternatives, the solidtumor is selected from the group consisting of a breast cancer, lungcancer, colon cancer, renal cancer, pancreatic cancer, prostate cancer,and ovarian cancer.

In some alternatives, a method of performing cellular immunotherapy in asubject having cancer or a viral infection is provided wherein themethod comprises administering a composition or a host cell to thesubject and administering the drug or metabolite thereof, therebyinducing expression from the promoter. In some alternatives, the hostcell is a primary human lymphocyte, wherein the host cell comprises a) anucleic acid comprising a first promoter, which is an induciblesynthetic promoter containing a binding site for a DNA binding domainnot naturally present in the primary human lymphocyte and b) apolynucleotide coding for a transcriptional activator, thetranscriptional activator comprising i) the DNA binding domain, whereinthe DNA binding domain does not specifically bind to a DNA sequencenaturally present in the primary human lymphocyte; ii) a domain thatspecifically binds to a drug or metabolite thereof and does not bind ordoes not bind with as great a degree of affinity to any moleculenaturally present in the primary human lymphocyte and iii) atransactivation domain, wherein the transcriptional activator is capableof inducing transcription from the first promoter in the presence of thedrug or metabolite thereof and/or upon binding of the drug or metabolitethereof to the domain in (ii). In some alternatives, the cell furthercomprises a polynucleotide encoding a chimeric antigen receptor, whichoptionally is operably linked to the first promoter. In somealternatives, In some alternatives, the cell further comprises a secondpromoter, which is operably linked to the polynucleotide encoding thetranscriptional activator, wherein the second promoter is optionallyconstitutive. In some alternatives, the host cell is a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cell. In some alternatives, the host cell is a CD4+ T helperlymphocyte cell that is selected from the group consisting of naïve CD4+T cells, central memory CD4+ T cells, effector memory CD4+ T cells, andbulk CD4+ T cells. In some alternatives, the composition comprises ahost cell in a pharmaceutically acceptable excipient. In somealternatives, the host cell the host cell is a primary human lymphocyte,wherein the host cell comprises a) a nucleic acid comprising a firstpromoter, which is an inducible synthetic promoter containing a bindingsite for a DNA binding domain not naturally present in the primary humanlymphocyte and b) a polynucleotide coding for a transcriptionalactivator, the transcriptional activator comprising i) the DNA bindingdomain, wherein the DNA binding domain does not specifically bind to aDNA sequence naturally present in the primary human lymphocyte; ii) adomain that specifically binds to a drug or metabolite thereof and doesnot bind or does not bind with as great a degree of affinity to anymolecule naturally present in the primary human lymphocyte and iii) atransactivation domain, wherein the transcriptional activator is capableof inducing transcription from the first promoter in the presence of thedrug or metabolite thereof and/or upon binding of the drug or metabolitethereof to the domain in (ii). In some alternatives, the cell furthercomprises a polynucleotide encoding a chimeric antigen receptor, whichoptionally is operably linked to the first promoter. In somealternatives, the cell further comprises a second promoter, which isoperably linked to the polynucleotide encoding the transcriptionalactivator, wherein the second promoter is optionally constitutive. Insome alternatives, the host cell is a CD8+ T cytotoxic lymphocyte cellselected from the group consisting of naïve CD8+ T cells, central memoryCD8+ T cells, effector memory CD8+ T cells and bulk CD8+ T cell. In somealternatives, the host cell is a CD4+ T helper lymphocyte cell that isselected from the group consisting of naïve CD4+ T cells, central memoryCD4+ T cells, effector memory CD4+ T cells, and bulk CD4+ T cells. Insome alternatives, the composition comprises a CD8+ T cytotoxiclymphocyte cell selected from the group consisting of naïve CD8+ Tcells, central memory CD8+ T cells, effector memory CD8+ T cells andbulk CD8+ T cell and a CD4+ T helper lymphocyte cell that is selectedfrom the group consisting of naïve CD4+ T cells, central memory CD4+ Tcells, effector memory CD4+ T cells, and bulk CD4+ T cells. In somealternatives, the cancer is selected from a solid tumor or hematologicmalignancy. In some alternatives, the solid tumor is selected from thegroup consisting of a breast cancer, lung cancer, colon cancer, renalcancer, pancreatic cancer, prostate cancer, and ovarian cancer.

Another aspect of the disclosure includes a genetic system to deliverdrug-regulated transgene expression in cells, such as drug-regulatedexpression of a recombinant protein such as a recombinant antigenreceptor and/or a molecule expressed by a cell expressing a recombinantantigen receptor. In an alternative, regulated transgene expression isengineered into and/or contained within cells, such as lymphocytes, forexample, for use in adoptive cell therapy, such as adoptiveimmunotherapy. Such systems provide rigorous safety attributes to celltherapies, such as chimeric antigen receptor (CAR) adoptive therapeuticstrategies, generally without sacrificing curative intent. In someaspects, such features permit real-time clinician control of recombinantprotein expression, e.g., CAR expression, in vivo. By engineeringvectors that enable drug responsive transcriptional control ofrecombinant gene, e.g., CAR, expression, the activity of the recombinantgene, e.g., CARs, and/or other cell mediators can be turned “ON” and“OFF” in vivo, for example, based on a clinician prescribedpharmaceutical drug input that exhibits clinically permissivepharmacokinetics, tissue distribution, and partitioning between theextracellular space and cytosol of lymphocytes. The genetic systemprovides for drug regulated transgene expression to enforce a functional“OFF” state in the absence of the drug and a functional “ON” statetransgene expression in the presence of the drug.

One alternative of such a drug is tamoxifen. Tamoxifen is an estrogenantagonist/partial agonist that is an FDA-approved and commerciallyavailable drug. It is taken orally and can be administered on a dailybasis over an extended period of time. Tamoxifen has a proven safetyrecord, favorable pharmacokinetic profile, excellent tissue distributionand a low partition coefficient between the extracellular space andcytosol. Functional analogs of tamoxifen also may be used. Other drugscan be selected, for example, based on safety record, favorablepharmacokinetic profile, excellent tissue distribution, a low partitioncoefficient between the extracellular space and cytosol, and/or lowtoxicities.

In some alternatives, the system employs a synthetic transcriptionalregulator, e.g., synthetic transcriptional activator, which, in thepresence of tamoxifen, can be induced to bind to a synthetic promoteroperably linked to, e.g., upstream of, a transgene, to induce expressionfrom the promoter, e.g., of the transgene. In some alternatives providedherein, the transcriptional activator is regulated by a drug ormetabolite thereof, such as a tamoxifen-regulated transcriptionalactivator or transcription factor, which may be regulated, e.g.,induced, by tamoxifen or an analog or metabolite thereof. Exemplarytamoxifen-regulated transcription factors are chimeric transcriptionfactors, such as those comprising a DNA binding domain specific for asynthetic promoter of the system, a domain that specifically bindstamoxifen and/or metabolite(s) thereof, for example, with affinityhigher than the affinity of the domain for a natural molecule such asestrogen, and a transactivating domain, such as a strong transactivatingdomain. One tamoxifen regulated transcription factor (“TamR-tf”, alsodesignated “HEA3”) is a chimeric transcription factor composed of humansubunits including the N-terminal DNA binding domain of HepatocyteNuclear Factor 1-alpha (HNF-1α) fused in frame to a mutant (G521R)tamoxifen-specific form of an estrogen receptor ligand binding domain(ER-LBD), which binds to tamoxifen metabolites with high affinity ascompared to estrogen, which is in turn fused to the p65 activationdomain of NF-κB (p65). An exemplary amino acid sequence of a TamR-tf isprovided in FIGS. 9A, 9B, and 9C and is identified as SEQ ID NO: 40. Inthis sequence, the mutant tamoxifen-specific form of the ligand bindingdomain of the estrogen receptor ligand binding domain (ER-LBD) is foundat amino acids 282-595 of the TamR-tf and has a mutation at position521, as compared to wild-type estrogen receptor ligand binding domain.The p65 activation domain of NF-κB (p65) is found at amino acids 596-862of SEQ ID NO: 40. Changes can be made to the transcriptional activator,for example, to increase the properties of the transcription factorincluding, without limitation, altering one or more amino acids in theestrogen receptor ligand binding domain to increase the affinity of thefactor for estrogen analogs and altering one or more amino acids in thep65 transactivating domain. In some alternatives, changes are made tothe transcriptional activator that result in an altered transcriptionfactor which retains or substantially retains one or more functions oftamR-tf, such as tamoxifen-specific binding and/or the same orsubstantially the same or at least the same specificity for tamoxifen ormetabolite as compared to any natural molecule, at least the same orabout the same specific DNA binding function, and/or the same or atleast the same degree of transactivation activity.

In the absence of tamoxifen, the transcriptional activator, e.g.,TamR-tf, is generally excluded from the nucleus by binding of cytosolicheat-shock protein 90 (HSP90) to the tamoxifen-binding active site,resulting in the expression of a transgene operably linked to thetamR-tf-inducible promoter being in the “OFF” state. Nanomolarconcentrations of cytosolic tamoxifen actively out-compete HSP90 forER-LBD binding, resulting in TamR-tf translocation to the nucleus. Uponnuclear translocation, TamR-tf is readily available to bind to arestricted synthetic promoter (e.g. 7xHBD/EF1αp). In the presence oftamoxifen, binding of TamR-tf to the synthetic promoter, e.g., the7xHBD/EF1αp promoter, induces the “ON” state of expression for atransgene operably linked to the synthetic promoter. In somealternatives, this transcriptional regulator can be modified to providefor a varying level of control of transgene expression. Amino acidsubstitutions in the LBD of TamR-tf permit selective responsiveness totamoxifen and its metabolites, where 4-hydroxy tamoxifen (4-OHT) is themost pharmacologically active metabolite, in regards to TamR-tfactivity, while lacking interaction with endogenous estrogen. In somealternatives, a system for inducible expression of chimeric antigenreceptor is provided, wherein the system comprises: a first nucleic acidcomprising a first promoter inducible by a drug operably linked to apolynucleotide, such as one coding for a chimeric antigen receptor, thechimeric antigen receptor optionally comprising a ligand binding domain,wherein the ligand binding domain binds to a ligand, wherein the ligandis a tumor specific molecule, viral molecule, or any other moleculeexpressed on a target cell population that is suitable to mediaterecognition and elimination by a lymphocyte, a polypeptide spacer,wherein the spacer optionally provides for increased T cellproliferation and/or cytokine production in response to the ligand ascompared to a reference chimeric receptor, a transmembrane domain, andan intracellular signaling domain. In some alternatives, the systemfurther includes a second nucleic acid encoding a transcriptionalmodulator for the inducible promoter, which is capable of modulating,e.g., activating, transcription from the first promoter, such as in thepresence of the drug or metabolite thereof; typically the systemcomprises a second constitutive or inducible promoter operably linked toa nucleic acid coding for the transcriptional modulator.

In some alternatives of the inducible system, the first promoter isoperably linked to a polynucleotide coding for a gene that promotes cellsurvival and proliferation, a gene that prevents apoptosis, and/or agene that that inhibits negative checkpoint signaling. Such genesinclude genes encoding IL-2, IL-15, Chemokine receptors, Bcl2, CA-Akt,dn-TGFbetaRIII, dn-SHP1/2, and/or PD-1CD28 chimeras.

In some alternatives, the system employs a synthetic transcriptionalactivator which, in the presence of the drug (e.g.tamoxifen) or ametabolite thereof, such as following administration of the drug, isinduced to bind a synthetic promoter upstream of a transgene to induceexpression. In some alternatives, the transcriptional activator isTamR-tf (HEA3) or other tamoxifen-inducible transcriptional activatorwith analogous domains. The tamoxifen regulated transcription factor(“TamR-tf”, also designated “HEA3”) is a chimeric transcription factorcomposed of human subunits including the N-terminal DNA binding domainof Hepatocyte Nuclear Factor 1-alpha (HNF-1α) (e.g. amino acids 1-281 ofSEQ ID NO: 40) fused in frame to the mutant tamoxifen-specific ligandbinding domain of the estrogen receptor ligand binding domain (ER-LBD),that is in turn fused to the p65 activation domain of NF-κB (p65).

In some alternatives of the compositions herein, the CD4+ T helperlymphocyte cell is naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells, or bulk CD4+ T cells. In somealternatives, CD4+ helper lymphocyte cell is a naïve CD4+ T cell,wherein the naïve CD4+ T cell comprises a CD45RO−, CD45RA+, and/or is aCD62L+ CD4+ T cell. In some alternatives, at least 20, 30, 40, 50, 60,70, 80, 90, 95, 99, or 100% of the cells in the composition are CD4+; insome alternatives, at least 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or100% of the cells in the composition or the CD4+ cells in thecomposition are naïve CD4+ T cells, central memory CD4+ T cells,effector memory CD4+ T cells, or bulk CD4+ T cells.

In some alternatives of the compositions herein, the CD8+ T cytotoxiclymphocyte cell is a naïve CD8+ T cell, central memory CD8+ T cell,effector memory CD8+ T cell and/or bulk CD8+ T cell. In somealternatives, the CD8+ cytotoxic T lymphocyte cell is a central memory Tcell, wherein the central memory T cell comprises a CD45RO+, CD62L+,and/or CD8+ T cell. In yet other alternatives, the CD8+ cytotoxic Tlymphocyte cell is a central memory T cell and the CD4+ helper Tlymphocyte cell is a naïve or central memory CD4+ T cell. In somealternatives, at least 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100%of the cells in the composition are CD8+; in some alternatives, at least20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100% of the cells in thecomposition or the CD8+ cells in the composition are naïve CD8+ T cells,central memory CD8+ T cells, effector memory CD8+ T cells, or bulk CD8+T cells. In some embodiments, at least 20, 30, 40, 50, 60, 70, 80, 90,95, 99, or 100% of the cells in the composition, and/or of the CD4+and/or CD8+ cells in the composition, express the transgene orrecombinant molecule, such as the CAR, and/or contain the expressionsystem.

Additionally provided are methods of making compositions includingadoptive immunotherapy compositions, such as those containing thesystems, and uses or methods of using these compositions, such as forperforming cellular immunotherapy in a subject having a disease ordisorder.

In some alternatives, a method of manufacturing the compositionscomprises obtaining generating a modified naïve or naïve-derived orcentral memory or central memory-derived CD4+ T helper cell orpopulation containing the same, wherein the modified helper T lymphocytecell preparation comprises CD4+ T cells that have a chimeric receptor,such as one comprising a ligand binding domain specific for a tumor cellsurface molecule, a spacer domain, a transmembrane domain, and anintracellular signaling domain under control of an inducible promoter asdescribed herein. In other some alternatives, CD4+ cells have a cytokineor chemokine receptor under the control of an inducible promoter.

In some alternatives of the methods described herein, the T-cells areadministered with other T-cells that are non-CAR expressing, and/orproduce Tam-inducible proteins. In some alternatives, thetranscriptional activator for the CAR comprises a DNA-binding domain, atamofexin/metabolite binding domain, or a transactivation domain. Insome alternatives, one or more, generally all, of the domains from thesynthetic transcriptional activator is or are derived from humanproteins, such as human or substantially human domains. Such featurescan reduce immunogenicity of the constructs upon administration to humansubjects, for example, in cell therapy.

Expression in Jurkat Cells.

The “ON” and “OFF” state of Jurkat T cells expressing TamR ZsGreen wasstudied.

Constructs.

In some alternatives, this system involves two components: 1)constitutive expression of HEA-3 linked to a single transgene or set oftransgenes by T2A skip-linker domains and 2) conditional expression of atransgene that is under control of the HEA-3 restricted syntheticpromoter 7xHBD/mEF1αp whereby induction of the transgene occurs inresponse to tamoxifen. Depending on the desired use of TamRLV geneticcontrol, a combination of delivery systems and vector compositions canbe used. Construction of constructs is described above.

An alternative of the TamR-LV system allows for constitutive HEA3expression and inducible expression of either ZsGreen or chimericantigen receptors, permitting kinetic analysis and biological effectcontrolled in an “ON” and “OFF” manner by the presence or absence oftamoxifen. For this, two constructs were used: 1) HEA-3 is driven by thehuman EF1 a promoter and linked to the constructed truncated EGFR(EGFRt) transmembrane marker protein by a T2A skip-linker sequence andcloned into a third generation epHIV7 self-inactivating lentiviruspackaging plasmid (construct A). (See FIGS. 9A, 9B and 9C; SEQ ID NO:39; Table 1: SEQ ID NO: 8 and SEQ ID NO: 9) and 2) 7xHBD/mEF1αp controlstamoxifen-dependent expression of ZsGreen cloned into pcDNA3.1(−) whichwas engineered to lack the commercially used CMV promoter (construct B)(See Table 12; SEQ ID NO: 41). ZsGreen1 is a human codon-optimizedZsGreen variant that encodes the brightest commercially available greenfluorescent protein. (Available from Clontech).

Methods.

Jurkat cells were transduced at a MOI of 5 with lentivirus packagingconstructs described above in which a dual packaging approach in whicheach plasmid is co-transfected into 293T cells during lentiviralproduction. This construct utilized molar ratio of 1:2 of construct A toconstruct B. Construct A encodes the chimeric transcription factor HEA3linked via a ribosomal cleavage sequence, T2A, to the tracking andselection marker EGFRt. Construct B encodes the synthetic,HEA3-responsive promoter, 7xHBD/mE1b which regulates expression of thetransgene ZsGreen-DR1, a short half-life green fluorescent reportergene.

After transduction, cells were expanded in culture, enriched for EGFRtexpression utilizing Miltenyi magnetic bead selection, to a purity >99%.After further expansion in culture, cells were harvested and treatedwith Ethanol (Vehicle) as a negative control or 500 nM4-hydroxytamoxifen (40HT) for 24 hours, and then were harvested, stainedwith Erbitux-biotin followed by SA-APC, and analyzed for APC expressionand ZsGreen expression via flow cytometry. The results are shown in FIG.1.

Dose Response.

Cells were harvested and were then subjected to treatment with 4OHT withconcentrations ranging from 0 nM-1000 nM, as indicated. Samples wereharvested, washed, and stained with EGFRt-biotin, followed bystrepavidin-APC, and then analyzed by flow cytometry for ZsGreenexpression. The results are shown in FIG. 2.

On and Off Rate Kinetics.

The population was subjected to 500 nM 4OHT stimulation for 48 hours,and then sorted for ZsGreen+cells utilizing FACS, yielding a >99%ZsGreen+population on immediate post-sort analysis. Cells weresubsequently expanded in culture for 3 weeks, and then prepared forkinetic studies. Cells were divided into 3 treatment groups: (1) no 4OHT(2) 200 nM 4OHT (added every 48 hours) (3) 24 hour 200 nM 4OHT, followedby washout 1× in PBS, then culture in 4OHT free media for 14 days,followed by a 24 hour re-stimulation with 200 nm 4OHT, and then washoutin 1× PBS. At each time point, samples were harvested and analyzed viaflow cytometry for ZsGreen expression. Results are shown in FIG. 3 andare presented as % ZsGreen+.

Results.

The results in FIG. 1 show that in the presence of 4-hydroxy tamoxifen(4-OHT), about 50% of the cells express ZsGreen, indicating these cellshave both constructs A and B and that 4-OHT induced expression ofconstruct B. The results of the dose response curve show that aconcentration of 200 nM or greater of 4-OHT was effective to induceexpression of the transgene in transduced cells. (FIG. 2). The resultsin FIG. 3 show that as the 4-OHT is washed out of the culture, the ZsGreen expression drops off to less than 10% of the max ZsGReen activitywithin 5 days. When 4-OHT is added back, ZsGreen activity returns toabout 100% max activity within 2 days.

Discussion.

These experiments show that human T Jurkat cells can be transduced witha dual packaged lentivirus with a constitutive component and aninducible component. Expression of the ZsGreen gene is induced in thepresence of 4-OHT in a dose responsive manner. In addition, washing outof 4-OHT from the cell culture resulted in a decrease in expression ofZsGreen that could be re-stimulated by addition of 4-OHT back to thecells.

Expression in Primary CD4 Central Memory Cells and CD8 Central MemoryCells.

“ON” and “OFF” state of CD4 and CD8 Central memory T cells expressingTamR ZsGreen was studied.

Constructs.

In some alternatives, this system involves two components: 1)constitutive expression of HEA-3 linked to a single transgene or set oftransgenes by T2A skip-linker domains (construct A) (See FIGS. 9A, 9Band 9C; SEQ ID NO: 39; Table 1 SEQ ID NO: 8 and SEQ ID NO: 9) and 2)conditional expression of a transgene that is under control of the HEA-3restricted synthetic promoter 7xHBD/mEF1αp whereby induction of thetransgene occurs in response to tamoxifen (construct B) (See Table 12;SEQ ID NO: 41). Depending on the desired use of TamRLV genetic control acombination of delivery systems and vector compositions can be used.Constructs were prepared as described above.

Methods.

CD4 central memory cells were obtained from peripheral blood byselecting cells through flow cytometry for markers CD4, and CD62L andnegative for CD45RO. Cells were cultured with anti-CD3/anti-CD28 beadsfor 3 days.

CD8 central memory cells were obtained from peripheral blood byselecting cells through flow cytometry for markers CD8, and CD62L andnegative for CD45RO. Cells were cultured with anti-CD3/anti-CD28 beadsfor 3 days.

After 3 days, CD4 or CD8 central memory cells were transduced at a MOIof 5 with lentivirus packaging constructs described above in which adual packaging approach in which each plasmid is co-transfected into293T cells during lentiviral production. This construct utilized molarratio of 1:2 of construct A to construct B. Construct A encodes thechimeric transcription factor HEA3 linked via a ribosomal cleavagesequence, T2A, to the tracking and selection marker EGFRt. Construct Bencodes the synthetic, HEA3-responsive promoter, 7xHBD/mE1b whichregulates expression of the transgene ZsGreen-DR1, a short half-lifegreen fluorescent reporter gene.

On day 21 after transduction, cells were enriched for EGFRt, andsubsequently expanded with feeder cells, IL2, and IL15. Followingexpansion, cells were divided into 3 treatment groups, 4OHT alone (A),4OHT combined with CD3/CD28 bead cotreatment (B) or 4OHT alone for 48hours, followed by addition of CD3/CD28 beads (C). Corresponding sampleswithout 4OHT were also obtained for comparison. All samples wereharvested at indicated time points following 4OHT treatment, stainedwith EGFRt-biotin antibody, followed by SA-APC, and analyzed for flowcytometry. The results are shown in FIG. 4 (CD4 central memory) and FIG.5 (CD8 central memory).

Results.

The results show that the CD4 cells transduced with the dual plasmidvector required the presence of activation stimulus in order to expressthe ZsGreen even in the presence of tamoxifen. See FIG. 4B. About 70% ofthe primary transduced CD4 cells in the presence of tamoxifen andantiCD3/anti-CD28 beads expressed ZsGreen. Gene expression was seen whenactivation occurred after a 48 hour treatment with tamoxifen. See FIG.4C.

The results show that the CD8 cells transduced with the dual plasmidvector also required the presence of activation stimulus in order toexpress the ZsGreen even in the presence of tamoxifen. See FIG. 5B.About 37% of the primary transduced CD8 cells in the presence oftamoxifen and antiCD3/anti-CD28 beads expressed ZsGreen. Gene expressionwas seen when activation occurred after a 48 hour treatment withtamoxifen. See FIG. 5C.

These results show that primary central memory T cells transduced withan inducible construct can express the transgene in the presence of theinducer upon activation with antiCD3/CD28 cells. However, nonactivatedcells expressing the transgene can be readily isolated usingimmunomagnetic or flow cytometry sorting.

Construction of TamR—CD19CAR LV.

Construction of the vector was accomplished by dual packaging oftransfer plasmids housing constructs as described above at a plasmidmolar ratio of 1:1. (See FIGS. 6C and 7C) Construct A under theconstitutive EF-lalpha promoter encodes TamR-tf (HEA3) linked via aribosomal cleavage sequence, T2A, to the tracking and selection markerEGFRt. (See FIGS. 9A, 9B and 9C; SEQ ID NO: 39, Table 1: SEQ ID NO: 8and SEQ ID NO: 9) Construct B contains the 7xHBD/mE1b promoter withdownstream transgene cDNA including CD19CAR linked via 2a cleavagesequence to Her2t, a tracking and selection marker. (See Table 12; SEQID NO: 41; Table 2 SEQ ID NO: 10; FIG. 11; SEQ ID NO: 44). Anotherconstruct was prepared adding an additional selective marker DHFRdm (SeeFIG. 12; SEQ ID NO: 46).

Human Jurkat cells were transduced at a MOI of 2, then were expanded inculture, and selected for EGFRt+ cells via magnetic bead selection.After further expansion, cells were either treated with vehicle alone(EtOH) or 500 nM 4OHT, and harvested for flow cytometry. Samples werestained with EGFRt-APC antibody and Herceptin-biotin, followed by SA PE.Samples were also prepared for western blot, the primary antibody usedis an anti-CD247 mouse mAb which recognizes the intracellular CD3 zetachain on the CD19CAR (˜48 kDa). The endogenous CD3 zeta migrates at ˜23kDa. The results are shown in FIG. 6.

Jurkat T Cells Transduced with TamR CD19CAR LV Containing a SelectiveMarker.

Construction of TamR CD19CAR LV was done through dual packaging ofconstructs A and B. Construct A contains HEA3 linked via a ribosomalcleavage sequence, T2A, to the tracking and selection marker EGFRt.Construct B contains the 7xHBD/mE1b promoter with downstream transgeneconsisting of CD19CAR linked via 2a cleavage sequence to Her2t, atracking and selection marker, linked via another 2a cleavage sequence(P2A) to DHFRdm, a methotrexate selection gene.

Cells were transduced with TamR CD19CAR LV at a MOI of 1, expanded inculture, selected for EGFRt via magnetic bead selection. Followingfurther expansion cells were treated with vector alone (EtOH) or 500 nM4OHT, and harvested for flow cytometry, (upper panel). FIG. 7. Sampleswere stained with EGFRt-APC antibody and Herceptin-biotin, followed bySA-PE. Parental (untransduced Jurkat) is shown for comparison. Sampleswere also prepared for western blot, the primary antibody used is ananti-CD247 mouse mAb which recognizes the intracellular CD3 zeta chainon the CD19CAR (˜48 kDa). The endogenous CD3 zeta migrates at ˜23 kDa.

Results.

The results show that EGFRt is detected in 94.6% of cells transducedwith a construct not including the additional selective marker DHFRdm.See FIG. 6B. FIG. 6C shows that in the presence of tamoxifen, about42.6% of the cells transduced with a construct not including DHFRdmexpressed both EGFRt and Her2t. The cells expressing both markersexpressed the CD3zeta chain as part of the CAR construct (48 kDa) asdetected in Western blot. See FIG. 6B.

FIG. 7 shows that 85.9% Jurkat cells transduced with a constructincluding another 2a cleavage sequence (P2A) to DHFRdm in the absence oftamoxifen express EGFRt. In the presence of tamoxifen, about 7.5% of thecells expressed both EGFRt and Her2t. The cells expressing both markersexpressed the CD3zeta chain as part of the CAR construct (48 kDa) asdetected in Western blot. See FIG. 7B.

Inducible expression of CD19 CAR is shown in transduced Jurkat cells.Induction can be measured by detecting expression of the marker Her2t aswell as detecting expression of CD19CAR using western blot. Adding anadditional selectable marker, DHFRdm, to the inducible construct did notimprove inducible gene expression.

TamR CD19CAR LV Transduced Human CD4 TCM T Cells.

CD4 central memory cells were obtained from peripheral blood byselecting cells through flow cytometry for markers CD4, and CD62L andnegative for CD45RO. Cells were cultured anti-CD3/anti-CD28 beads for 3days. After 3 days, CD4 Central memory cells were transduced at a MOI of5 with a lentivirus which packages constructs A and B. (FIG. 8B).Construct A encodes the chimeric transcription factor HEA3 linked via aribosomal cleavage sequence, T2A, to the tracking and selection markerEGFRt. (See FIGS. 9A, 9B and 9C; SEQ ID NO: 39; Table 1; SEQ ID NO: 8and SEQ ID NO: 9). Construct B encodes the synthetic, HEA3-responsivepromoter, 7xHBD/mE1b which regulates expression of the transgeneCD19CAR-linked via a T2A to Her2t for tracking and selection, and via asecond 2a sequence, P2A, to the methotrexate selection gene DHFRdm. (SeeTable 12; SEQ ID NO: 41; Table 2 SEQ ID NO: 10; FIG. 11; SEQ ID NO: 44;FIG. 12; SEQ ID NO: 46).

On day 21 after transduction, cells were enriched for EGFRt utilizingmagnetic bead selection, and subsequently expanded with irradiatedfeeder cells, IL2, and IL15. Two weeks after expansion, cells werecryopreserved. In the experiment depicted above, CD4+ CEM cells whichwere mock transduced, or CD4+TamR CD19CAR LV were thawed, placed inculture with CD3/CD28 beads, IL-2, IL-15. CD4+TamRCD19CAR LV weretreated or not with 500 nM 4OHT for 24 hours. All samples were harvestedwashed, then stained with an EGFRt-APC antibody and Her2t-biotin,followed by SA-PE, and analyzed via flow cytometry. The results areshown in FIG. 8.

Results.

The results in FIG. 8 show that in the absence of tamoxifen 75.6% oftransduced primary CD4 central memory cells express EGFRt, indicatingthe constitutive expression of construct A. In the presence of tamoxifenand an activating stimulus (e.g. antiCD3/antiCD28 beads), about 14.7% ofthe primary cells express both EGFRt and Her2t.

Primary CD4 cells transduced with an inducible vector in the presence ofthe inducer and an activating stimulus express the transgene undercontrol of the inducible promoter as detected by the expression of themarker gene Her2t. However, nonactivated cells expressing the transgenecan be readily isolated using immunomagnetic or flow cytometry sorting.Further characterization of these cells will involve detecting ofexpression of CD19CAR.

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein. Allreferences and documents referred to herein are hereby incorporated byreference.

TABLE 1 Sequence of anti-CD19 short spacer chimeric receptorGMCSFRss-CD19scFv-IgG4hinge- CD28tm-41BB-Zeta-T2A-EGFRt (GMCSFRss)(SEQ ID NO: 2) Atg ctgctgctggtgaccagcctgctgctgtgcgagctgccccaccccgcctttctgctgatcccc (CD19scFv) (SEQ ID NO: 3)Gacatccagatgacccagaccacctccagcctgagcgccagcctgggcgaccgggtgaccatcagctgccgggccagccaggacatcagcaagtacctgaactggtatcagcagaagcccgacggcaccgtcaagctgctgatctaccacaccagccggctgcacagcggcgtgcccagccggtttagcggcagcggctccggcaccgactacagcctgaccatctccaacctggaacaggaagatatcgccacctacttttgccagcagggcaacacactgccctacacctttggcggcggaacaaagctggaaatcaccggcagcacctccggcagcggcaagcctggcagcggcgagggcagcaccaagggcgaggtgaagctgcaggaaagcggccctggcctggtggcccccagccagagcctgagcgtgacctgcaccgtgagcggcgtgagcctgcccgactacggcgtgagctggatccggcagccccccaggaagggcctggaatggctgggcgtgatctggggcagcgagaccacctactacaacagcgccctgaagagccggctgaccatcatcaaggacaacagcaagagccaggtgttcctgaagatgaacagcctgcagaccgacgacaccgccatctactactgcgccaagcactactactacggcggcagctacgccatggactactggggccagggcaccagcgtgaccgtgagcagc (IgG4hinge)(SEQ ID NO: 4) Gaatctaagtacggaccgccctgccccccttgccct (CD28tm-)(SEQ ID NO: 5) Atgttctgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcttttgggtg (41BB) (SEQ ID NO: 6)Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg (CD3Zeta)- (SEQ ID NO: 7)Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcagaagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccaggaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcccccaagg (T2A) (SEQ ID NO: 8)Ctcgagggcggcggagagggcagaggaagtcttctaacatgcggtgacgtggaggagaatcccggccctagg (EGFRt) (SEQ ID NO: 9)Atgcttctcctggtgacaagccactgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcctgccggtggcatttaggggtgactccttcacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcagttttctcttgcagtcgtcagcctgaacataacatccttgggattacgctccctcaaggagataagtgatggagatgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaattataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccttctggagggtgagccaagggagtttgtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccctggggatcggcctcttcatg tga

TABLE 2     GMCSFRss DNA: ATGCTGCTGCTGGTGACCAGCCTGCTGCTGTGCGAGCTGCCCCACCCCGCC AA:M  L  L  L  V  T  S  L  L  L  C  E  L  P  H  P  A                  CD19scFv DNA:TTTCTGCTGATCCCC:GACATCCAGATGACCCAGACCACCTCCAGCCTGAGC AA:F  L  L  I  P   D  I  Q  M  T  Q  T  T  S  S  L  S DNA:GCCAGCCTGGGCGACCGGGTGACCATCAGCTGCCGGGCCAGCCAGGACATC AA:A  S  L  G  D  R  V  T  I  S  C  R  A  S  Q  D  I DNA:AGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTCAAGCTG AA:S  K  Y  L  N  W  Y  Q  Q  K  P  D  G  T  V  K  L DNA:CTGATCTACCACACCAGCCGGCTGCACAGCGGCGTGCCCAGCCGGTTTAGC AA:L  I  Y  H  T  S  R  L  H  S  G  V  P  S  R  F  S DNA:GGCAGCGGCTCCGGCACCGACTACAGCCTGACCATCTCCAACCTGGAACAG AA:G  S  G  S  G  T  D  Y  S  L  T  I  S  N  L  E  Q DNA:GAAGATATCGCCACCTACTTTTGCCAGCAGGGCAACACACTGCCCTACACC AA:E  D  I  A  T  Y  F  C  Q  Q  G  N  T  L  P  Y  T DNA:TTTGGCGGCGGAACAAAGCTGGAAATCACCGGCAGCACCTCCGGCAGCGGC AA:F  G  G  G  T  K  L  E  I  T  G  S  T  S  G  S  G DNA:AAGCCTGGCAGCGGCGAGGGCAGCACCAAGGGCGAGGTGAAGCTGCAGGAA AA:K  P  G  S  G  E  G  S  T  K  G  E  V  K  L  Q  E DNA:AGCGGCCCTGGCCTGGTGGCCCCCAGCCAGAGCCTGAGCGTGACCTGCACC AA:S  G  P  G  L  V  A  P  S  Q  S  L  S  V  T  C  T DNA:GTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCCGGCAGCCC AA:V  S  G  V  S  L  P  D  Y  G  V  S  W  I  R  Q  P DNA:CCCAGGAAGGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCGAGACCACC AA:P  R  K  G  L  E  W  L  G  V  I  W  G  S  E  T  T DNA:TACTACAACAGCGCCCTGAAGAGCCGGCTGACCATCATCAAGGACAACAGC AA:Y  Y  N  S  A  L  K  S  R  L  T  I  I  K  D  N  S DNA:AAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCC AA:K  S  Q  V  F  L  K  M  N  S  L  Q  T  D  D  T  A DNA:ATCTACTACTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGAC AA:I  Y  Y  C  A  K  H  Y  Y  Y  G  G  S  Y  A  M  D                                         IgG4hinge DNA:TACTGGGGCCAGGGCACCAGCGTGACCGTGAGCAGC:GAGAGCAAGTACGGA AA:Y  W  G  Q  G  T  S  V  T  V  S  S   E  S  K  Y  G                          CD28tm DNA:CCGCCCTGCCCCCCTTGCCCT:ATGTTCTGGGTGCTGGTGGTGGTCGGAGGC AA:P  P  C  P  P  C  P   M  F  W  V  L  V  V  V  G  G DNA:GTGCTGGCCTGCTACAGCCTGCTGGTCACCGTGGCCTTCATCATCTTTTGG AA:V  L  A  C  Y  S  L  L  V  T  V  A  F  I  I  F  W     41B16 DNA:GTG:AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATG AA:V   K  R  G  R  K  K  L  L  Y  I  F  K  Q  P  F  M DNA:AGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCA AA:R  P  V  Q  T  T  Q  E  E  D  G  C  S  C  R  F  P                                         CD3Zeta DNA:GAAGAAGAAGAAGGAGGATGTGAACTGCGGGTGAAG:TTCAGCAGAAGCGCC AA:E  E  E  E  G  G  C  E  L  R  V  K   F  S  R  S  A DNA:GACGCCCCTGCCTACCAGCAGGGCCAGAATCAGCTGTACAACGAGCTGAAC AA:D  A  P  A  Y  Q  Q  G  Q  N  Q  L  Y  N  E  L  N DNA:CTGGGCAGAAGGGAAGAGTACGACGTCCTGGATAAGCGGAGAGGCCGGGAC AA:L  G  R  R  E  E  Y  D  V  L  D  K  R  R  G  R  D DNA:CCTGAGATGGGCGGCAAGCCTCGGCGGAAGAACCCCCAGGAAGGCCTGTAT AA:P  E  M  G  G  K  P  R  R  K  N  P  Q  E  G  L  Y DNA:AACGAACTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATCGGCATG AA:N  E  L  Q  K  D  K  M  A  E  A  Y  S  E  I  G  M DNA:AAGGGCGAGCGGAGGCGGGGCAAGGGCCACGACGGCCTGTATCAGGGCCTG AA:K  G  E  R  R  R  G  K  G  H  D  G  L  Y  Q  G  L DNA:TCCACCGCCACCAAGGATACCTACGACGCCCTGCACATGCAGGCCCTGCCC AA:S  T  A  T  K  D  T  Y  D  A  L  H  M  Q  A  L  P           T2A DNA:CCAAGG:CTCGAGGGCGGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGT AA:P  R   L  E  G  G  G  E  G  R  G  S  L  L  T  C  G                                     EGFRt DNA:GACGTGGAGGAGAATCCCGGCCCTAGG:ATGCTTCTCCTGGTGACAAGCCTT AA:D  V  E  E  N  P  G  P  R   M  L  L  L  V  T  S  L DNA:CTGCTCTGTGAGTTACCACACCCAGCATTCCTCCTGATCCCACGCAAAGTG AA:L  L  C  E  L  P  H  P  A  F  L  L  I  P  R  K  V DNA:TGTAACGGAATAGGTATTGGTGAATTTAAAGACTCACTCTCCATAAATGCT AA:C  N  G  I  G  I  G  E  F  K  D  S  L  S  I  N  A DNA:ACGAATATTAAACACTTCAAAAACTGCACCTCCATCAGTGGCGATCTCCAC AA:T  N  I  K  H  F  K  N  C  T  S  I  S  G  D  L  H DNA:ATCCTGCCGGTGGCATTTAGGGGTGACTCCTTCACACATACTCCTCCTCTG AA:I  L  P  V  A  F  R  G  D  S  F  T  H  T  P  P  L DNA:GATCCACAGGAACTGGATATTCTGAAAACCGTAAAGGAAATCACAGGGTTT AA:D  P  Q  E  L  D  I  L  K  T  V  K  E  I  T  G  F DNA:TTGCTGATTCAGGCTTGGCCTGAAAACAGGACGGACCTCCATGCCTTTGAG AA:L  L  I  Q  A  W  P  E  N  R  T  D  L  H  A  F  E DNA:AACCTAGAAATCATACGCGGCAGGACCAAGCAACATGGTCAGTTTTCTCTT AA:N  L  E  I  I  R  G  R  T  K  Q  H  G  Q  F  S  L DNA:GCAGTCGTCAGCCTGAACATAACATCCTTGGGATTACGCTCCCTCAAGGAG AA:A  V  V  S  L  N  I  T  S  L  G  L  R  S  L  K  E DNA:ATAAGTGATGGAGATGTGATAATTTCAGGAAACAAAAATTTGTGCTATGCA AA:I  S  D  G  D  V  I  I  S  G  N  K  N  L  C  Y  A DNA:AATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAA AA:N  T  I  N  W  K  K  L  F  G  T  S  G  Q  K  T  K DNA:ATTATAAGCAACAGAGGTGAAAACAGCTGCAAGGCCACAGGCCAGGTCTGC AA:I  I  S  N  R  G  E  N  S  C  K  A  T  G  Q  V  C DNA:CATGCCTTGTGCTCCCCCGAGGGCTGCTGGGGCCCGGAGCCCAGGGACTGC AA:H  A  L  C  S  P  E  G  C  W  G  P  E  P  R  D  C DNA:GTCTCTTGCCGGAATGTCAGCCGAGGCAGGGAATGCGTGGACAAGTGCAAC AA:V  S  C  R  N  V  S  R  G  R  E  C  V  D  K  C  N DNA:CTTCTGGAGGGTGAGCCAAGGGAGTTTGTGGAGAACTCTGAGTGCATACAG AA:L  L  E  G  E  P  R  E  F  V  E  N  S  E  C  I  Q DNA:TGCCACCCAGAGTGCCTGCCTCAGGCCATGAACATCACCTGCACAGGACGG AA:C  H  P  E  C  L  P  Q  A  M  N  I  T  C  T  G  R DNA:GGACCAGACAACTGTATCCAGTGTGCCCACTACATTGACGGCCCCCACTGC AA:G  P  D  N  C  I  Q  C  A  H  Y  I  D  G  P  H  C DNA:GTCAAGACCTGCCCGGCAGGAGTCATGGGAGAAAACAACACCCTGGTCTGG AA:V  K  T  C  P  A  G  V  M  G  E  N  N  T  L  V  W DNA:AAGTACGCAGACGCCGGCCATGTGTGCCACCTGTGCCATCCAAACTGCACC AA:K  Y  A  D  A  G  H  V  C  H  L  C  H  P  N  C  T DNA:TACGGATGCACTGGGCCAGGTCTTGAAGGCTGTCCAACGAATGGGCCTAAG AA:Y  G  C  T  G  P  G  L  E  G  C  P  T  N  G  P  K DNA:ATCCCGTCCATCGCCACTGGGATGGTGGGGGCCCTCCTCTTGCTGCTGGTG AA:I  P  S  I  A  T  G  M  V  G  A  L  L  L  L  L  V DNA:GTGGCCCTGGGGATCGGCCTCTTCATG

 (SEQ ID NO: 10) AA: V  A  L  G  I  G  L  F  M  *   (SEQ ID NO: 11)

TABLE 3 ZXR-014 Nucleotide and amino acid sequences (map of sections)GMCSFRss: nt2084-2149 (SEQ ID NO: 50)CD19scFv: nt2150-2884(SEQ ID NO: 51)Igg4Hinge: nt2885-2920 (SEQ ID NO: 52)CD28tm: nt2921-3004 (SEQ ID NO: 53) 41BB: nt3005-3130 (SEQ ID NO: 54)Zeta: nt3131-3466 (SEQ ID NO: 55) T2A: nt3467-3538 (SEQ ID NO: 56)EGFRt: nt3539-4612 (SEQ ID NO: 57) Primers for sequencing: Oligo nameSequence Region oJ02649 ATCAAAAGAATAG pre-U5 ACCGAGATAGGGT(SEQ ID NO: 22) oJ02648 CCGTACCTTTAAG delU3 ACCAATGACTTAC(SEQ ID NO: 23) oJ02650 TTGAGAGTTTTCG mid-Ampr CCCCG (SEQ ID NO: 24)oJ02651 AATAGACAGATCG post-Ampr CTGAGATAGGT (SEQ ID NO: 25) oJ02652CAGGTATCCGGTA CoE1 ori AGCGG (SEQ ID NO: 26) oJ02653 CGACCAGCAACCA SV40TAGTCC (SEQ ID NO: 27) oJ02654 TAGCGGTTTGACT CMV CACGG (SEQ ID NO: 28)oJ02655 GCAGGGAGCTAGA psi ACGATTC (SEQ ID NO: 29) oJ02656 ATTGTCTGGTATARRE GTGCAGCAG (SEQ ID NO: 30) oJ02657 TCGCAACGGGTTT EF1p GCC(SEQ ID NO: 31) oJ02658 AGGAAGATATCGC CD19Rop CACCTACT (SEQ ID NO: 32)oJ02601 CGGGTGAAGTTCA Zeta GCAGAAG (SEQ ID NO: 33) oJ02735 ACTGTGTTTGCTGWPRE ACGCAAC (SEQ ID NO: 34) oJ02715 ATGCTTCTCCTGG EGFRt TGACAAG(SEQ ID NO: 35)

TABLE 4 Uniprot P0861 IgG4-Fc (SEQ ID NO: 13)        10         20         30         40 ASTKGPSVFP LAPCSRSTSE STAALGCLVK DYFPEPVTVS        50         60         70         80 WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTKT        90        100        110        120YTCNVDHKPS NTKVDKRVES KYGPPCPSCP APEFLGGPSV       130        140        150        160 FLFPPKPKDT LMISRTPEVT CVVVDVSQED PEVQFNWYVD       170        180        190        200 GVEVHNAKTK PREEQFNSTY RVVSVLTVLH QDWLNGKEYK       210        220        230        240CKVSNKGLPS SIEKTISKAK GQPREPQVYT LPPSQEEMTK       250        260        270        280 NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS       290        300        310        320DGSFFLYSRL TVDKSRWQEG NVFSCSVMHE ALHNHYTQKS LSLSLGK1-98  CH1 (SEQ ID NO: 59) 99-110 Hinge (SEQ ID NO: 60)111-220 CH2 (SEQ ID NO: 61) 221-327 CH3 (SEQ ID NO: 62)Position 108 S→P (SEQ ID NO: 63)

TABLE 5 Uniprot P10747 CD28 (SEQ ID NO: 14)        10         20         30         40MLRLLLALNL FPSIQVTGNK ILVKQSPMLV AYDNAVNLSC        50         60         70         80 KYSYNLFSRE FRASLHKGLD SAVEVCVVYG NYSQQLQVYS        90        100        110        120KTGFNCDGKL GNESVTFYLQ NLYVNQTDIY FCKIEVMYPP       130        140        150        160 PYLDNEKSNG TIIHVKGKHL CPSPLFPGPS KPFWVLVVVG       170        180        190        200 GVLACYSLLV TVAFIIFWVR SKRSRLLHSD YMNMTPRRPG        210        220PTRKHYQPYA PPRDFAAYRS 1-18 signal peptide (SEQ ID NO: 64)19-152 extracellular domain (SEQ ID NO: 65)153-179 transmembrane domain (SEQ ID NO: 66)180-220 intracellular domain (SEQ ID NO: 67)Position 186-187 LL→GG (SEQ ID NO: 68)

TABLE 6 Uniprot Q07011 4-1BB (SEQ ID NO: 15)        10         20         30         40MGNSCYNIVA TLLLVLNFER TRSLQDPCSN CPAGTFCDNN        50         60         70         80 RNQICSPCPP NSFSSAGGQR TCDICRQCKG VFRTRKECSS        90        100        110        120TSNAECDCTP GFHCLGAGCS MCEQDCKQGQ ELTKKGCKDC       130        140        150        160 CFGTFNDQKR GICRPWTNCS LDGKSVLVNG TKERDVVCGP       170        180        190        200 SPADLSPGAS SVTPPAPARE PGHSPQIISF FLALTSTALL       210        220        230        240FLLFFLTLRF SVVKRGRKKL LYIFKQPFMR PVQTTQEEDG        250 CSCRFPEEEE GGCEL1-23 signal peptide (SEQ ID NO: 69)24-186 extracellular domain (SEQ ID NO: 70)187-213 transmembrane domain (SEQ ID NO: 71)214-255 intracellular domain (SEQ ID NO: 72)

TABLE 7 Uniprot P20963 human CD3ζ Isoform 3 (SEQ ID NO: 16)        10         20         30         40MKWKALFTAA ILQAQLPITE AQSFGLLDPK LCYLLDGILF        50         60         70         80 IYGVILTALF LRVKFSRSAD APAYQQGQNQ LYNELNLGRR        90        100        110        120EEYDVLDKRR GRDPEMGGKP QRRKNPQEGL YNELQKDKMA       130        140        150        160 EAYSEIGMKG ERRRGKGHDG LYQGLSTATK DTYDALHMQA LPPR1-21 signal peptide (SEQ ID NO: 73) 22-30 extracellular (SEQ ID NO: 74)31-51 transmembrane (SEQ ID NO: 75)52-164 intracellular domain (SEQ ID NO: 76) 61-89 ITAM1 (SEQ ID NO: 77)100-128 ITAM2 (SEQ ID NO: 78) 131-159 ITAM3 (SEQ ID NO: 79)

TABLE 8 Exemplary Hinge region Sequences Human IgG1 (SEQ ID NO: 17)EPKSCDKTHTCPPCP Human IgG2 (SEQ ID NO: 18) ERKCCVECPPCP Human IgG3(SEQ ID NO: 19) ELKTPLGDTHTCPRCP (EPKSCDTPPPCPRCP)₃ Human IgG4(SEQ ID NO: 20) ESKYGPPCPSCP Modified Human IgG4 (SEQ ID NO: 21)ESKYGPPCPPCP Modified Human IgG4 (SEQ ID NO: 36) YGPPCPPCPModified Human IgG4 (SEQ ID NO: 37) KYGPPCPPCP Modified Human IgG4(SEQ ID NO: 38) EVVKYGPPCPPCP

TABLE 9 Her 2 construct-short spacer (SEQ ID NO: 1) GMCSFss-Her2scFv-IgG4hinge-CD28tm-41BB-Zeta-T2A-EGFRt Leader (SEQ ID NO: 80)Atgcttctcctggtgacaagccactgctctgtgagttaccacaccca gcattcctcctgatcccaHer2scFV (SEQ ID NO: 81) Gatatccagatgacccagtccccgagctccctgtccgcctctgtgggcgatagggtcaccatcacctgccgtgccagtcaggatgtgaatactgctgtagcctggtatcaacagaaaccaggaaaagctccgaaactactgatttactcggcatccacctctactctggagtcccactcgcttctctggaccagatctgggacggatttcactctgaccatcagcagtctgcagccggaagacttcgcaacttattactgtcagcaacattatactactcctcccacgttcggacagggtaccaaggtggagatcaaaggcagtactagcggcggtggctccgggggcggatccggtgggggcggcagcagcgaggttcagctggtggagtctggcggtggcctggtgcagccagggggctcactccgtagtcctgtgcagcactggcttcaacattaaagacacctatatacactgggtgcgtcaggccccgggtaagggcctggaatgggagcaaggatttatcctacgaatggttatactagatatgccgatagcgtcaagggccctcactataagcgcagacacatccaaaaacacagcctacctgcagatgaacagcctgcgtgctgaggacactgccgtctattattgttctagatggggaggggacggcttctatgctatggactactggggtcaagg aaccctggtcaccgtctcgagtHinge spacer (SEQ ID NO: 82) Gagagcaagtacggaccgccctgccccccttgccct CD28tm (SEQ ID NO: 83) Atgactgggtgctggtggtggtcggaggcgtgctggcctgctacagcctgctggtcaccgtggccttcatcatcattgggtg 4-1BB (SEQ ID NO: 84)Aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg CD3 zeta (SEQ ID NO: 85)Cgggtgaagttcagcagaagcgccgacgcccctgcctaccagcagggccagaatcagctgtacaacgagctgaacctgggcagaagggaagagtacgacgtcctggataagcggagaggccgggaccctgagatgggcggcaagcctcggcggaagaacccccaggaaggcctgtataacgaactgcagaaagacaagatggccgaggcctacagcgagatcggcatgaagggcgagcggaggcggggcaagggccacgacggcctgtatcagggcctgtccaccgccaccaaggatacctacgacgccctgcacatgcaggccctgcc cccaagg T2A(SEQ ID NO: 86) Ctcgagggcggcggagagggcagaggaagtatctaacatgcggtgacgtggaggagaatcccggccctagg tEGFR (SEQ ID NO: 87)atgcttctcctggtgacaagccactgctctgtgagttaccacacccagcattcctcctgatcccacgcaaagtgtgtaacggaataggtattggtgaatttaaagactcactctccataaatgctacgaatattaaacacttcaaaaactgcacctccatcagtggcgatctccacatcctgccggtggcatttaggggtgactccacacacatactcctcctctggatccacaggaactggatattctgaaaaccgtaaaggaaatcacagggtttttgctgattcaggcttggcctgaaaacaggacggacctccatgcctttgagaacctagaaatcatacgcggcaggaccaagcaacatggtcagattctcttgcagtcgtcagcctgaacataacatccagggattacgctccctcaaggagataagtgatggagatgtgataatttcaggaaacaaaaatttgtgctatgcaaatacaataaactggaaaaaactgtttgggacctccggtcagaaaaccaaaattataagcaacagaggtgaaaacagctgcaaggccacaggccaggtctgccatgccttgtgctcccccgagggctgctggggcccggagcccagggactgcgtctcttgccggaatgtcagccgaggcagggaatgcgtggacaagtgcaaccactggagggtgagccaagggagtagtggagaactctgagtgcatacagtgccacccagagtgcctgcctcaggccatgaacatcacctgcacaggacggggaccagacaactgtatccagtgtgcccactacattgacggcccccactgcgtcaagacctgcccggcaggagtcatgggagaaaacaacaccctggtctggaagtacgcagacgccggccatgtgtgccacctgtgccatccaaactgcacctacggatgcactgggccaggtcttgaaggctgtccaacgaatgggcctaagatcccgtccatcgccactgggatggtgggggccctcctcttgctgctggtggtggccctggggatcggcctcttcatgtga

TABLE 12 7xHBD/mEF1α nucleic acid sequence (SEQ ID NO: 41)Tagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataatctacaatagttaataat ctacaatagttaataatctacaa

1.-45. (canceled)
 46. A cell comprising: a first nucleic acid comprisingan inducible first promoter comprising a plurality of HNF-1 bindingsites and an albumin promoter TATA box, wherein the first nucleic acidis operably linked to a polynucleotide encoding a chimeric antigenreceptor (CAR), wherein the CAR comprises: a ligand binding domaincapable of specifically binding to a ligand, a spacer providing anincreased activity for the CAR in the cell to induce T cellproliferation or cytokine production in response to the CAR binding theligand compared to the activity of a reference CAR, a transmembranedomain, and an intracellular signaling domain; and a second nucleic acidcomprising a second promoter operably linked to a nucleic acid encodinga transactivator for the first promoter.
 47. The cell of claim 46,wherein the first promoter is inducible with tamoxifen or with ametabolite of tamoxifen.
 48. The cell of claim 47, wherein the firstpromoter comprises a sequence having at least 95% identity with thenucleic acid sequence of SEQ ID NO:41.
 49. The cell of claim 48, whereinthe first promoter comprises a sequence having the nucleic acid sequenceof SEQ ID NO:41.
 50. The cell of claim 46, wherein the second promoteris an EF1αp promoter.
 51. The cell of claim 46, wherein thetransactivator comprises an HEA-3 polypeptide.
 52. The cell of claim 51,wherein the transactivator comprises a polypeptide having at least 95%identity with the amino acid sequence of SEQ ID NO:40.
 53. The cell ofclaim 52, wherein the transactivator comprises a polypeptide having theamino acid sequence of SEQ ID NO:40.
 54. The cell of claim 46, wherein:the ligand binding domain is capable of specifically binding to CD19 orHer2; the spacer is an IgG4 hinge spacer; the transmembrane domaincomprises a CD28 transmembrane domain; or the intracellular signalingdomain comprises an 41BB domain and/or a CD3 zeta domain.
 55. The cellof claim 54, wherein: the ligand binding domain encoded by a nucleicacid having at least 95% identity with the nucleic acid sequence of SEQID NO:03 or SEQ ID NO:81; the spacer encoded by a nucleic acid having atleast 95% identity with the nucleic acid sequence of SEQ ID NO:04; thetransmembrane domain encoded by a nucleic acid having at least 95%identity with the nucleic acid sequence of SEQ ID NO:05; or theintracellular signaling domain comprising a domain encoded by a nucleicacid having at least 95% identity with the nucleic acid sequence of SEQID NO:06 and/or SEQ ID NO:07.
 56. The cell of claim 46, wherein the cellis a T cell.
 57. The cell of claim 56, wherein the cell is a CD4+ T cellor a CD8+ T cell.
 58. The cell of claim 57, wherein the cell is a CD8+ Tcytotoxic lymphocyte cell selected from the group consisting of a naïveCD8+ T cell, central memory CD8+ T cell, effector memory CD8+ T cell andbulk CD8+ T cells.
 59. The cell of claim 57, wherein the cell is a CD4+T helper lymphocyte cell selected from the group consisting of a naïveCD4+ T cell, central memory CD4+ T cell, effector memory CD4+ T cell,and bulk CD4+ T cell.
 60. The cell of claim 46, wherein the cell is aprecursor T cell.
 61. The cell of claim 60, wherein the cell is ahematopoietic stem cell.
 62. The cell of claim 46, wherein the cell isex vivo.
 63. A pharmaceutical composition comprising the cell of claim46 and a pharmaceutically acceptable excipient.
 64. A compositioncomprising: the cell of claim 46 contacted with an activating stimulatorselected from anti-CD3 or an anti-CD28.
 65. The composition of claim 64,wherein the cell is contacted with tamoxifen or with a metabolite oftamoxifen.